Method for delivering bioactive agents into and through the mucosally-associated lymphoid tissue and controlling their release

ABSTRACT

A method, and compositions for use therein capable, of delivering a bioactive agent to an animal entailing the steps of encapsulating effective amounts of the agent in a biocompatible excipient to form microcapsules having a size less than approximately ten micrometers and administering effective amounts of the microcapsules to the animal. A pulsatile response is obtained, as well as mucosal and systemic immunity.

This invention was made with government support under Contract No.DAMD17-86-C-6162 awarded by the Department of the Army of the UnitedStates Government. The U.S. Government has certain non-commercial rightsin the invention. The U.S. Government does not have rights in theinvention pertaining to drug delivery.

REFERENCE TO CO-PENDING APPLICATION

This application is a continuation of application Ser. No. 08/116,484,filed Sep. 7, 1993, which is a continuation of application Ser. No.07/629,138, filed Dec. 18, 1990, now abandoned, which is acontinuation-in-part of application Ser. No. 07/325,193, filed Mar. 16,1989, now abandoned, which is a continuation-in-part of application Ser.No. 07/169,973, filed Mar. 18, 1988, now U.S. Pat. No. 5,075,109, whichis a continuation-in-part of application Ser. No. 06/923,159, filed Oct.24, 1986, now abandoned.

TABLE OF CONTENTS

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE FIGURES

DETAILED DESCRIPTION OF THE INVENTION

I. MICROENCAPSULATION

A. Preparation of Dye-Loaded Microcapsules.

B. Preparation of Antigen-Loaded Microcapsules.

II. PENETRATION OF DYE-LOADED MICROCAPSULES INTO THE PEYER'S PATCHESAFTER ORAL ADMINISTRATION

EXAMPLE 1--Polystyrene Microcapsules.

EXAMPLE 2--85:15 Poly(DL-lactide-co-glycolide) Microcapsules.

1. Uptake of Biocompatible and Biodegradable Microcapsules into thePeyer's Patches.

2. Microcapsule Migration to the Mesenteric Lymph Nodes and Spleen.

EXAMPLE 3--Comparison of the Uptake of Microcapsules of 10 Compositionsby the Peyer's Patches.

III. ANTIBODY RESPONSES INDUCED WITH MICROENCAPSULATED VACCINES

MATERIALS AND METHODS

Trinitrophenyl--Keyhole Limpet Hemocyanin.

Immunizations.

Collection of Biological Fluids.

1. Plasma.

2. Intestinal Secretions.

3. Saliva.

4. Bronchial-Alveolar Wash Fluids.

5. Immunochemical Reagents.

6. Solid-Phase Radioimmunoassays.

A. Vaccine-Microcapsules Administered by Injection.

1. Adjuvant Effect Imparted by Microencapsulation.

EXAMPLE 1--Adjuvant Effect Imparted byMicroencapsulation-Intraperitoneal Administration.

EXAMPLE 2--Adjuvant Effect Imparted by Microencapsulation-SubcutaneousAdministration.

2. Mechanism of the Adjuvant Effect Imparted by Microencapsulation.

EXAMPLE 1--The Adjuvant Effect Imparted by Microencapsulation is Not theResult of Adjuvant Activity Intrinsic to the Polymer.

EXAMPLE 2--Retarding the Antigen Release Rate from 1-10 MicrometerMicrocapsules Increases the Level of the Antibody Response and Delaysthe Time of the Peak Response.

EXAMPLE 3--Correlation of the Size of the Microcapsules with theResultant Adjuvant Effect.

3. Pulsatile Release of Vaccines from Microcapsules for ProgrammedBoosting Following a Single Injection

EXAMPLE 1--Co-administration of Free and Microencapsulated Vaccine.

EXAMPLE 2--Co-administration of <10 Micrometer Priming and >10Micrometer Pulsing Vaccine Microcapsules.

EXAMPLE 3--Co-administration of <10 Micrometer Priming and <10Micrometer Pulsing Vaccine Microcapsules.

B. Vaccine-Microcapsules Administered Orally

EXAMPLE 1--Orally Administered Microspheres Containing TNP-KLH InduceConcurrent Circulating and Mucosal Antibody Responses to TNP.

EXAMPLE 2--Orally Administered Microcapsules Containing SEB ToxoidInduce Concurrent Circulating and Mucosal Anti-SEB Toxin Antibodies.

C. Vaccine Microcapsules Administered Intratracheally.

EXAMPLE 1--Intratracheally Administered Microcapsules Containing SEBToxoid Induce Concurrent Circulating and Mucosal Anti-Toxin Antibodies.

D. Vaccine Microcapsules Administered by Mixed Immunization Routes.

IV. ABSORPTION OF PHARMACEUTICALS.

BACKGROUND OF THE INVENTION

This invention relates to a method and a formulation for orallyadministering a bioactive agent encapsulated in one or morebiocompatible polymer or copolymer excipients, preferably abiodegradable polymer or copolymer, affording microcapsules which due totheir proper size and physical chemical properties results in themicrocapsules and contained agent reaching and being effectively takenup by the folliculi lymphatic aggregati, otherwise known as the "Peyer'spatches", of the gastrointestinal tract in an animal without loss ofeffectiveness due to the agent having passed through thegastrointestinal tract. Similar folliculi lymphatic aggregati can befound in the respiratory tract, genitourinary tract, large intestine andother mucosal tissues of the body such as ophthalmic tissues. Hereafter,the above-described tissues are referred to in general asmucosally-associated lymphoid tissues.

The use of microencapsulation to protect sensitive bioactive agents fromdegradation has become well-known. Typically, a bioactive agent isencapsulated within any of a number of protective wall materials,usually polymeric in nature. The agent to be encapsulated can be coatedwith a single wall of polymeric material (microcapsules), or can behomogeneously dispersed within a polymeric matrix (microspheres).(Hereafter, the term microcapsules refers to both microcapsules andmicrospheres). The amount of agent inside the microcapsule can be variedas desired, ranging from either a small amount to as high as 95% or moreof the microcapsule composition. The diameter of the microcapsule canalso be varied as desired, ranging from less than one micrometer to aslarge as three millimeters or more.

Peyer's patches are aggregates of lymphoid nodules located in the wallof the small intestine, large intestine and appendix and are animportant part of body's defense against the adherence and penetrationof infectious agents and other substances foreign to the body. Antigensare substances that induce the antibody-producing and/or cell-mediatedimmune systems of the body, and include such things as foreign proteinor tissue. The immunologic response induced by the interaction of anantigen with the immune system may be either positive or negative withrespect to the body's ability to mount an antibody or cell-mediatedimmune response to a subsequent reexposure to the antigen. Cell-mediatedimmune responses include responses such as the killing of foreign cellsor tissues, "cell-mediated cytoxicity", and delayed-typehypersensitivity reactions. Antibodies belong to a class of proteinscalled immunoglobulins (Ig), which are produced in response to anantigen, and which combine specifically with the antigen. When anantibody and antigen combine, they form a complex. This complex may aidin the clearance of the antigen from the body, facilitate the killing ofliving antigens such as infectious agents and foreign tissues orcancers, and neutralize the activity of toxins or enzymes. In the caseof the mucosal surfaces of the body the major class of antibody presentin the secretions which bathe these sites is secretory immunoglobulin A(sIgA). Secretory IgA antibodies prevent the adherence and penetrationof infectious agents and other antigens to and through the mucosaltissues of the body.

While numerous antigens enter the body through the mucosal tissues,commonly employed immunization methods, such as intramuscular orsubcutaneous injection of antigens or vaccines, rarely induce theappearance of sIgA antibodies in mucosal secretions. Secretory IgAantibodies are most effectively induced through direct immunization ofthe mucosally-associated lymphoid tissues, of which the Peyer's patchesof the gastrointestinal tract represent the largest mass in the body.

Peyer's patches possess IgA precursor B cells which can populate thelamina propria regions of the gastrointestinal and upper respiratorytracts and differentiate into mature IgA synthesizing plasma cells. Itis these plasma cells which actually secrete the antibody molecules.Studies by Heremans and Bazin measuring the development of IgA responsesin mice orally immunized with antigen showed that a sequentialappearance of antigen-specific IgA plasma cells occurred, first inmesenteric lymph nodes, later in the spleen, and finally in the laminapropria of the gastrointestinal tract (Bazin, H., Levi, G., and Doria,G. Predominant contribution of IgA antibody-forming cells to an immuneresponse detected in extraintestinal lymphoid tissues of germ free miceexposed to antigen via the oral route. J. Immunol. 105:1049; 1970 andCrabbe, P. A., Nash, D. R., Bazin, H., Eyssen, H. and Heremans, J. F.Antibodies of the IgA type in intestinal plasma cells of germ-free miceafter oral or parenteral immunization with ferritin. J. Exp. Med.130:723; 1969). Subsequent studies have shown that oral administrationof antigens leads to the production of sIgA antibodies in the gut andalso in mucosal secretions distant to the gut, e.g., in bronchialwashings, colostrum, milk, saliva and tears (Mestecky, J., McGhee, J.R., Arnold, R. R., Michalek, S. M., Prince, S. J. and Babb, J. L.Selective induction of an immune response in human external secretionsby ingestion of bacterial antigen. J. Clin. Invest. 61:731; 1978,Montgomery, P. C., Rosner, B. R. and Cohen, J. The secretory antibodyresponse. Anti-DNP antibodies induced by dinitrophenylated Type IIIpneumococcus. Immunol. Commun. 3:143; 1974, and Hanson, L. A., Ahistedt,S., Carlsson, B., Kaijser, B., Larsson, P., MattsbyBaltzer, A., SohlAkerlund, A., Svanborg Eden, C. and Dvennerholm, A. M. Secretory IgAantibodies to enterobacterial virulence antigens: their induction andpossible relevance, Adv. Exp. Med. Biol, 1007:165; 1978). It isapparent, therefore, that Peyer's patches are an enriched source ofprecursor IgA cells, which, subsequent to antigen sensitization, followa circular migrational pathway and account for the expression of IgA atboth the region of initial antigen exposure and at distant mucosalsurfaces. This circular pattern provides a mucosal immune system bycontinually transporting sensitized B cells to mucosal sites forresponses to gut-encountered environmental antigens and potentialpathogens.

Of particular importance to the present invention is the ability of oralimmunization to induce protective antibodies. It is known that theingestion of antigens by animals results in the appearance ofantigen-specific sIgA antibodies in bronchial and nasal washings. Forexample, studies with human volunteers show that oral administration ofinfluenza vaccine is effective at inducing secretory anti-influenzaantibodies in nasal secretions.

Extensive studies have demonstrated the feasibility of oral immunizationto induce the common mucosal immune system, but with rare exception thelarge doses require to achieve effective immunization have made thisapproach impractical. It is apparent that any method or formulationinvolving oral administration of an ingredient be of such design thatwill protect the agent from degradation during its passage through thegastrointestinal tract and target the delivery of the ingredient to thePeyer's patches. If not, the ingredient will reach the Peyer's patches,if at all, in an inadequate quantity or ineffective condition.

Therefore, there exists a need for a method of oral immunization whichwill effectively stimulate the immune system and overcome the problem ofdegradation of the antigen during its passage through thegastrointestinal tract to the Peyer's patch. There exists a moreparticular need for a method of targeting an antigen to the Peyer'spatches and releasing that antigen once inside the body. There alsoexists a need for a method to immunize through other mucosal tissues ofthe body which overcomes the problems of degradation of the antigen andtargets the delivery to the mucosally-associated lymphoid tissues. Inaddition, the need exists for the protection from degradation ofmucosally applied bioactive agents, improves and/or targets theirentrance into the body through the mucosally-associated lymphoid tissuesand releases the bioactive agent once it has entered the body.

SUMMARY OF THE INVENTION

This invention relates to a method and formulation for targeting to andthen releasing a bioactive agent in the body of an animal by mucosalapplication, and in particular, oral and intratracheal administration.The agent is microencapsulated in a biocompatible polymer or copolymer,preferably a biodegradable polymer or copolymer which is capable ofpassing through the gastrointestinal tract or existing on a mucosalsurface without degradation or with minimal degradation so that theagent reaches and enters the Peyer's patches or othermucosally-associated lymphoid tissues unaltered and in effectiveamounts. The term biocompatible is defined as a polymeric material whichis not toxic to the body, is not carcinogenic, and which should notinduce inflammation in body tissues. It is preferred that themicrocapsule polymeric excipient be biodegradable in the sense that itshould degrade by bodily processes to products readily disposable by thebody and should not accumulate in the body. The microcapsules are alsoof a size and physicalchemical composition capable of being effectivelyand selectively taken up by the Peyer's patches. Therefore, the problemsof the agent reaching the Peyer's patch or other mucosally-associatedtissue and being taken up are solved.

It is an object of this invention to provide a method of orallyadministering an antigen to an animal which results in the antigenreaching and being taken up by the Peyer's patches, and therebystimulating the mucosal immune system, without losing its effectivenessas a result of passing through the animal's gastrointestinal tract.

It is also an object of this invention to provide a method of orallyadministering an antigen to an animal which results in the antigenreaching and being taken up by the Peyer's patches, and therebystimulating the systemic immune system, without losing its effectivenessas a result of having passed through the gastrointestinal tract.

It is a further object of this invention to provide a method ofadministering an antigen to an animal which results in the antigenreaching and being taken up by the mucosally-associated lymphoidtissues, and thereby stimulating the mucosal immune system, withoutlosing its effectiveness as a result of degradation on the mucosalsurface.

It is a still further object of this invention to provide a method ofadministering an antigen to an animal which results in the antigen beingtaken up by the mucosally-associated lymphoid tissues, and therebystimulating the systemic immune system, without losing its effectivenessas-a result of degradation on the mucosal surface.

It is a still further object of this invention to provide a method oforally administering a bioactive agent to an animal which results in theagent reaching and being taken up by the Peyer's patches, and therebyresulting in an increased local or systemic concentration of the agent.

It is a still further object of this invention to provide a method ofadministering a bioactive agent to an animal which results in the agentreaching and being taken up by the mucosally-associated lymphoidtissues, and thereby resulting in an increased local or systemicconcentration of the agent.

It is a still further object of this invention to provide a formulationconsisting of a core bioactive ingredient and an encapsulating polymeror copolymer excipient which is biocompatible and preferablybiodegradable as well, which can be utilized in themucosal-administration methods described above.

It is another object of this invention to provide an improved vaccinedelivery system which obviates the need for immunopotentiators.

It is a still further object of this invention to provide an improvedvaccine delivery system for the induction of immunity through thepulsatile release of antigen from a single administration ofmicroencapsulated antigen.

It is a still further object of this invention to provide an improvedvaccine delivery system which both obviates the need forimmunopotentiators and affords induction of immunity through pulsatilereleases of antigen all from a single administration of microcapsulatedantigen.

It is a further object of this invention to provide a compositioncapable of achieving these above-referenced objects

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the plasma IgG responses in mice followingsubcutaneous administration of 1-10 μm and 10-110 μm 85:15 DL-PLG SEBtoxoid-containing microspheres.

FIG. 2 represents the plasma IgG responses in mice determined byendpoint titration following subcutaneous administration of 1-10 μm,20-50 μm and a mixture of 1-10 and 20-50 μm SEB toxoid-containingmicrocapsules.

FIG. 3 represents the plasma IgG responses in mice followingsubcutaneous administration of 1-10 μm 50:50 DL-PLG, 85:15 DL-PLG, and100:0 L-PLG SEB toxoid-containing microcapsules.

FIG. 4 represents the plasma IgG responses in mice followingsubcutaneous administration of 1-10 μm 50:50 DL-PLG, 100:0 L-PLG, and amixture of 50:50 DL-PLG and 100:0 L-PLG SEB toxoid-containingmicrocapsules.

DETAILED DESCRIPTION OF THE INVENTION

Illustrations of the methods performing embodiments of the inventionfollow. These illustrations demonstrate the mucosally-associatedlymphoid tissue targeting and programmed delivery of the antigens(trinitrophenyl keyhole limpet hemocyanin and a toxoid vaccine ofstaphylococcal enterotoxin B), and a drug (etretinate) encapsulated in50:50 poly(DL-lactide-co-glycolide) to mice.

It should be noted, however, that other polymers besidespoly(DL-lactide-co-glycolide) may be used. Examples of such polymersinclude, but are not limited to, poly(glycolide),poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters andpoly(B-hydroxybutyric acid), and polyanhydrides.

Also, other bioactive ingredients may be used. Examples of such include,but are not limited to, antigens to vaccinate against viral, bacterial,protozoan, fungal diseases such as influenzae, respiratory syncytial,parainfluenza viruses, Hemophilus influenza, Bordetella pertussis,Neisseria gonorrhoeae, Streptococcus pneumoniae and Plasmodiumfalciparum or other diseases caused by pathogenic microorganisms orantigens to vaccinate against diseases caused by macroorganisms such ashelminthic pathogens or antigens to vaccinate against allergies.Additional bioactive agents which may be used included but are notlimited to, immunomodulators, nutrients, drugs, peptides, lymphokines,monokines and cytokines.

I. MICROENCAPSULATION

A. Preparation of Dye-Loaded Microcapsules

Coumarin, a water-insoluble fluorescent dye, was microencapsulated withpolystyrene, which is a nonbiodegradable polymer, to afford fluorescentmicrocapsules that could be used to follow the penetration ofmicrocapsules into the Peyer's patches. The procedure used to preparethese microcapsules follows:

First, a polymer solution is prepared by dissolving 4.95 g ofpolystyrene (Type 685D, Dow Chemical Company, Midland, Mich.) in 29.5 gof methylene chloride (Reagent Grade, Eastman Kodak, Rochester, N.Y.).Next, about 0.05 g of coumarin (Polysciences, Inc., Warrington, Pa.) isadded to the polymer solution and allowed to dissolve by stirring themixture with a magnetic stir bar.

In a separate container, 10 wt % aqueous poly(vinyl alcohol) (PVA)solution, the processing medium, is prepared by dissolving 40 g of PVA(Vinol 2050, Air Products and Chemicals, Allentown, Pa.) in 360 g ofdeionized water. After preparing the PVA solution, the solution issaturated by adding 6 g of methylene chloride. Next, the PVA solution isadded to a 1-L resin kettle (Ace Glass, Inc., Vineland, N.J.) fittedwith a truebore stir shaft and a 2.5-in. TEFLON impeller and stirred atabout 380 rpm by a Fisher stedi speed motor.

The polystyrene/coumarin mixture is then added to the resin kettlecontaining the PVA processing media. This is accomplished by pouring thepolystyrene/coumarin mixture through a long-stem 7-mm bore funnel whichdirects the mixture into the resin kettle. A stable oil-in-wateremulsion results and is subsequently stirred for about 30 minutes atambient pressure to afford oil microdroplets of the appropriate size.Then the resin kettle is closed, and the pressure in the resin kettle isgradually reduced to 520 mm Hg by means of a water aspirator connectedto a manometer and a bleed valve. The resin kettle contents are stirredat reduced pressure for about 24 hours to allow all of the methylenechloride to evaporate. After all of the methylene chloride hasevaporated, the hardened microcapsules are collected by centrifugationand dried for 72 hours in a vacuum chamber maintained at roomtemperature.

B. Preparation of Antigen-Loaded Microcapsules

TNP-KLH, a water-soluble antigen, was encapsulated inpoly(DL-lactide-co-glycolide), a biocompatible, biodegradable polyester.The procedure used to prepare the microcapsules follows:

First, a polymer solution was prepared by dissolving 0.5 g of 50:50poly(DL-lactide-co-glycolide) in 4.0 g of methylene chloride. Next, 300microliters of an aqueous solution of TNP-KLH (46 mg TNP-LKH/mL; afterdialysis) was added to and homogeneously dispersed in thepoly(DL-lactide-co-glycolide) solution by vortexing the mixture with aVortex-Genie 2 (Scientific Industries, Inc., Bohemia, N.Y.).

In a separate container, an 8 wt % aqueous PVA solution was prepared bydissolving 4.8 g of PVA in 55.2 g of deionized water. After dissolutionof the PVA, the PVA solution was added to a 100-mL resin kettle (KontesGlass, Inc., Vineland, N.J.) fitted with a truebore stirrer and a1.5-in. TEFLON turbine impeller. The polymer solution was then added tothe PVA processing medium by pouring through a long-stem 7-mm borefunnel. During this addition, the PVA solution was being stirred atabout 650 rpm. After the resulting oil-in-water emulsion was stirred inthe resin kettle for about 10 minutes, the contents of the resin kettlewere transferred to 3.5 L of deionized water contained in a 4-L beakerand being stirred at about 800 rpm with a 2-in. stainless steelimpeller. The resultant microcapsules were stirred in the deionizedwater for about 30 minutes, collected by centrifugation, washed twicewith deionized water to remove any residual PVA, and were then collectedby freeze drying. The microcapsule products consisted of sphericalparticles about 1 to 10 micrometers in diameter. Other microcapsules,such as staphylococcal enterotoxin B microcapsules, can be made in asimilar manner.

The TNP-KLH content of the antigen-loaded microcapsules, that is, thecore loading of the microcapsules, was determined by weighing out 10 mgof antigen-loaded microcapsules in a 12-mL centrifuge tube. Add 3.0 mLof methylene chloride to the tube and vortex to dissolve thepoly(DL-lactide-co-glycolide). Next, add 3.0 mL of deionized water tothe tube and vortex vigorously for 1 minute. Centrifuge the contents ofthe centrifuge tube to separate the organic and aqueous layers. Transferthe aqueous layer to a 10-mL volumetric flask. Repeat the extractioncombining the aqueous layers in the volumetric flask. Fill the flask tothe mark with deionized water. The amount of TNP-KLH in the flask, andsubsequently the amount of TNP-KLH in the microcapsules, is thenquantified using a protein assay. The microcapsules contained 0.2%TNP-KLH by weight. The staphylococcal enterotoxin B content ofstaphylococcal enterotoxin B microcapsules can be quantified in asimilar manner.

II. PENETRATION OF DYE-LOADED MICROCAPSULES INTO THE PEYER'S PATCHESAFTER ORAL ADMINISTRATION

By far the largest mass of tissue with the capacity to function as aninductive site for secretory IgA responses is the Peyer's patches. Thesediscrete nodules of lymphoreticular tissue are located along the entirelength of the small intestine and appendix. The targeted delivery ofintact antigen directly into this tissue to achieve high localconcentration is currently believed to be the most effective means ofinducing a disseminated mucosal IgA response. Biodegradablemicrocapsules represent an ideal vehicle to achieve this targetedvaccination.

EXAMPLE 1

Polystyrene Microcapsules

The uptake of microcapsules into the gut-associated lymphoreticulartissues and the size restriction of this penetration was investigated byorally administering to mice polystyrene microcapsules, loaded with thefluorescent dye coumarin. Unanesthetized, fasted BALB/c mice wereadministered 0.5 mL of a 100 mg/mL suspension of various sizedfluorescent microcapsules (less than 5 micrometers or 8 to 50micrometers in diameter) in tap water into the stomach using a feedingneedle. At various times after administration (0.5, 1 and 2 hours), themice were sacrificed and the small intestine excised. One-centimetersections of gut containing a discrete Peyer's patch were isolated,flushed of lumenal contents, everted and snap frozen. Frozen sectionswere prepared and examined under a fluorescence microscope to observethe number, location and size of the microcapsules which were taken upinto the Peyer's patch from the gut lumen.

Although some trapping of the microcapsules between the villi hadprevented their removal during flushing, no penetration into the tissueswas observed at any point except the Peyer's patch. At 0.5 hours afteroral administration, microcapsules were observed in the Peyer's patch ofthe proximal, but not the distal, portion of the small intestine., Withincreasing time the microcapsules were transported by peristalticmovement such that by 2 hours they were throughout the gastrointestinaltract and could be found in the Peyer's patch of the ilium. Theendocytosed microcapsules were predominantly located peripherally, awayfrom the apex of the Peyer's patch dome, giving the impression thatphysical trapping between the dome and adjacent villi during peristalsishad aided in their uptake. Comparison of the efficiency of uptake of the<5 micrometer versus the 8 to 50 micrometer preparations demonstratedthat microcapsules >10 micrometers in diameter were not absorbed intothe Peyer's patches while microcapsules of 1 to 10 micrometers indiameter were rapidly and selectively taken up. This suggested thatmicrocapsules composed of biodegradable wall materials would serve as aneffective means for the targeted delivery of antigens to thelymphoreticular tissues for the induction of immunity at mucosalsurfaces.

EXAMPLE 2

85:15 Poly(DL-lactide-co-glycolide) Microcapsules

1. Uptake of Biocompatible and Biodegradable Microcapsules into thePeyer's Patches

Groups of mice were administered biodegradable microcapsules containingthe fluorescent dye coumarin-6 as a suspension in tap water via agastric tube. The microcapsule wall material chosen for these studiesconsisted of 85:15 poly(DL-lactide-co-glycolide) due to its ability toresist significant bioerosion for a period of six weeks. At varioustimes from 1 to 35 days after administration, three representativePeyer's patches, the major mesenteric lymph nodes and the spleens fromindividual mice were removed, processed and serial frozen sectionsprepared.

When viewed with a fluorescence microscope using appropriate excitationand barrier filters the coumarin exhibited a deep green fluorescencewhich allowed the visual detection of microcapsules substantially lessthan 1 micrometer in diameter. All sections were viewed in order thatthe total number of microcapsules within each tissue or organ could bequantified. The size of each internalized microcapsule was determinedusing a calibrated eyepiece micrometer and its location within thetissue or organ was noted.

Internalized microcapsules of various sizes were observed in the Peyer'spatches at 24 hours post oral administration and at all time pointstested out to 35 days, as shown in Table 1. At no time weremicrocapsules of any size observed to penetrate into the tissue of thegut at any point other than the Peyer's patches. The total number ofmicrocapsules within the Peyer's patches increased through Day 4 andthen decreased over the following 31 days to approximately 15% of thepeak number.

This is consistent with the observation that free microcapsules could beobserved on the surface of the gut villi at the 1, 2 and 4 day timepoints. It is of interest that approximately 10 hours following oraladministration of the microcapsule suspension the coumarin-loadedmicrocapsules were frankly observable in the passed feces. Thisclearance was followed with the aid of an ultraviolet light source andby 24 hours the vast majority of the ingested microcapsules had beenpassed. Thus, the continued uptake of microcapsules into the Peyer'spatches observed at 2 and 4 days must be attributed to the minorfraction of the input dose which became entrapped within mucus betweenthe gut villi. In addition, the efficiency of uptake for the entrappedmicrocapsules must be several orders of magnitude greater than that ofthe microcapsules present in the gut lumen, but above the mucus layer.These observations are important when these data are extrapolated toman; the tremendously larger mass of Peyer's patch tissue and thegreatly increased transit time for the passage of material through thehuman small intestine relative to the mouse suggests that the efficiencyof microcapsule uptake into the human Peyer's patches will be muchhigher.

Microcapsules of various sizes were observed within the Peyer's patchesat all time points tested as shown in Table 1. At the 1, 2 and 4 daytime points the proportion of <2 micrometers (45-47%), 2-5 micrometers(31-35%) and >5 micrometers (18-23%) microcapsules remained relativelyconstant. Evident at 7 days, and even more so at later time points, wasa shift in the size distribution such that the small (<2 micrometers)and medium (2-5 micrometers) microcapsules ceased to predominate and thelarge (>5 micrometers) microcapsules became the numerically greatestspecies observed. This shift was concurrent with the decrease in totalmicrocapsule numbers in the Peyer's patches observed on and after Day 7.These results are consistent with the preferential migration of thesmall and medium sizes of microcapsules from the Peyer's patches whilethe large (>5 micrometers) microcapsules are preferentially retained.

Consistent with the preferential migration of the small and mediummicrocapsules out of the Peyer's patches are the data pertaining to thelocation of microcapsules within the architecture of the Peyer'spatches. When a microcapsule was observed within the Peyer's patch, itwas noted to be either relatively close to the dome epithelium where itentered the Peyer's patch (within 200 micrometers) or deeper within thelymphoid tissue (≧200 micrometers from the closest identifiable domeepithelium) (Table 1). Microcapsules observed deep within the Peyer'spatch tissue were almost exclusively of small and medium diameter. At 1day post-administration, 92% of the microcapsules were located close tothe dome epithelium. The proportion of deeply located microcapsulesincreased through Day 4 to 24% of the total, and thereafter decreasedwith time to approximately 2% at Day 14 and later. Thus, the small andmedium microcapsules migrate through and out of the Peyer's patches,while the large (>5 micrometers) microcapsules remain within the domeregion for an extended period of time.

2. Microcapsule Migration to the Mesenteric Lymph Nodes and Spleen

A small number of microcapsules were observed in the mesenteric lymphnodes at 1 day post-administration, and the numbers,progressivelyincreased through Day 7, as shown in Table 2. After Day 7, the numbersdecreased but were still detectable on Day 35. The size distributionclearly showed that microcapsules >5 micrometers in diameter did notenter this tissue, and the higher proportion of small (<2 micrometers)relative to medium (2-5 micrometers) microcapsules at the earlier timepoints indicated that the smaller diameter microcapsules migrate to thistissue with greatest efficiency. In addition, at the earlier timepoints, the majority of the microcapsules were located just under thecapsule in the subcapsular sinus. Later time points showed a shift inthe distribution to deep within the lymph node structure, and by day 14,90% of the microcapsules were located within the cortex and medullaryregions. The observation that the microcapsules are first detected in ornear the subcapsular sinus is consistent with their entry into thistissue via the lymphatics which drain the Peyer's patches. A progressiveincrease in the proportion of the microcapsules located deep in thistissue, clearly discernable at Day 4, followed by a progressive drop inthe total numbers on Day 14 and later, suggests that the microcapsulesprogress through this tissue and extravasate through the efferentlymphatic drainage.

Similar examination of the spleen showed that no microcapsules weredetectable until Day 4 post-administration. Peak numbers ofmicrocapsules were not observed in this organ until Day 14. As in thecase of the mesenteric lymph nodes, no microcapsules of >5 micrometersin diameter were observed. At all time points, the microcapsules wereobserved deep in this organ within the cortex. It should be noted thatthe peak number of microcapsules was observed in the spleen at a timewhen the majority of the microcapsules present in the mesenteric lymphnodes was deeply located and their total numbers falling. These data areconsistent with the known pattern of lymph drainage from the Peyer'spatches to the mesenteric lymph nodes and from the mesenteric lymphnodes to the bloodstream via the thoracic duct. Thus, it appears thatthe microcapsules present in the spleen have traversed the Peyer'spatches and mesenteric lymph nodes and have entered the spleen via theblood circulation.

In additional experiments, tissue sections from Peyer's patches,mesenteric lymph node and spleen which contained absorbed 85:15 DL-PLGmicrocapsules were examined by histochemical and immunohistochemicaltechniques. Among other observations, these studies clearly showed thatthe microcapsules which were absorbed into the Peyer's patches werepresent within macrophage-like cells which were stained by periodic acidSchiff's reagent (PAS) for intracellular carbohydrate, most probablyglycogen, and for major histocompatibility complex (MHC) class IIantigen. Further, the microcapsules observed in the mesenteric lymphnodes and in the spleen were universally found to have been carriedthere within these PAS and MHC class II positive cells. Thus, theantigen containing microcapsules have been internalized byantigen-presenting accessory cells (APC) in the Peyer's patches, andthese APC have disseminated the antigen-microcapsules to other lymphoidtissues.

These data indicate that the quality of the immune response induced byorally administering a microencapsulated vaccine can be controlled bythe size of the particles. Microcapsules <5 micrometers in diameterextravasate from the Peyer's patches within APC and release the antigenin lymphoid tissues which are inductive sites for systemic immuneresponses. In contrast, the microcapsules 5 to 10 micrometers indiameter remain in the Peyer's patches, also within APC, for extendedtime and release the antigen into this sIgA inductive site.

EXAMPLE 3

Comparison of the Uptake of Microcapsules of 10 Compositions by thePeyer's Patches

Experiments were performed to identify microcapsule polymeric excipientsthat would be useful for a practical controlled release delivery systemand which would possess the physicalchemical properties which wouldallow for targeted absorption of microcapsules into themucosally-associated lymphoid tissues. In regard to the latterconsideration, research has shown that hydrophobic particles are morereadily phagocytized by the cells of the reticuloendothelial system.Therefore, the absorption into the Peyer's patches of 1- to10-micrometer microcapsules of 10 different polymers which exhibit somerange with respect to hydrophobicity was examined. The wall materialschosen for these studies consisted of polymers that varied in wateruptake, biodegradation, and hydrophobicity. These polymers includedpolystyrene, poly(L-lactide), poly(DL-lactide), 50:50poly(DL-lactide-co-glycolide), 85:15 poly(DL-lactide-co-glycolide),poly(hydroxybutyric acid), poly(methyl methacrylate), ethyl cellulose,cellulose acetate hydrogen phthalate, and cellulose triacetate.Microcapsules, prepared from 7 of the 10 excipients, were absorbed andwere predominantly present in the dome region of the Peyer's patches 48hours after oral administration of a suspension containing 20 mg ofmicrocapsules, as shown in Table 3. None of the microspheres were seento penetrate into tissues other than the Peyer's patches. With oneexception, ethyl cellulose, the efficiency of absorption was found tocorrelate with the relative hydrophobicity of the excipient. Up to 1,500microcapsules were observed in the 3 representative Peyer's patches ofthe mice administered the most hydrophobic group of compoundspoly(styrene), poly(methyl methacrylate), poly(hydroxybutyrate)!, while200 to 1,000 microcapsules were observed with the relatively lesshydrophobic polyesters poly(L-lactide), poly(DL-lactide), 85:15poly(DL-lactide-co-glycolide), 50:50 poly(DL-lactide-co-glycolide)!. Asa class, the cellulosics were not absorbed.

It has been found that the physicalchemical characteristics of themicrocapsules regulate the targeting of the microcapsules through theefficiency of their absorption from the gut lumen by the Peyer'spatches, and that this is a surface phenomenon. Therefore, alterationsin the surface characteristics of the microcapsules, in the form ofchemical modifications of the polymer or in the form of coatings, can beused to regulate the efficiency with which the microcapsules target thedelivery of bioactive agents to mucosally-associated lymphoid tissuesand to APC. Examples of coatings which may be employed but are notlimited to, chemicals, polymers, antibodies, bioadhesives, proteins,peptides, carbohydrates, lectins and the like of both natural and manmade origin.

III. ANTIBODY RESPONSES INDUCED WITH MICROENCAPSULATED VACCINES

MATERIALS AND METHOD

Mice, BALB/c mice, 8 to 12 weeks of age, were used in these studies.

Trinitrophenyl--Keyhole Limpet Hemocyanin

Hemocyanin from the keyhole limpet (KLH) Megathura crenulate waspurchased from Calbiochem (San Diego, Calif.). It was conjugated withthe trinitrophenyl hapten (TNP-KLH) using 2, 4, 6-trinitrobenzenesulfonic acid according to the procedure of Rittenburg and Amkraut(Rittenburg, M. B. and Amkraut, A. A. Immunogenicity oftrinitrophenyl-hemocyanin: Production of primary and secondaryanti-hapten precipitins. J. Immunol. 97:421; 1966). The substitutionratio was spectrophotometrically determined to be TNP₈₆₁ -KLH using amolar extinction coefficient of 15,400 at a wavelength of 350 nm andapplying a 30% correction for the contribution of KLH at thiswavelength.

Staphylococcal Enterotoxin B Vaccine

A formalinized vaccine of staphylococcal enterotoxin B (SEB) wasprepared as described by Warren et al. (Warren, J. R., Spero, L. andMetzger, J. F. Antigenicity of formalin-inactivated staphylococcalenterotoxin B. J. Immunol. 111:885; 1973). In brief, 1 gm of enterotoxinwas dissolved in 0.1M sodium phosphate buffer, pH 7.5, to 2 mg/mL.Formaldehyde was added to the enterotoxin solution to achieve aformaldehyde:enterotoxin mole ratio of 4300:1. The solution was placedin a slowly shaking 37° C. controlled environment incubator-shaker andthe pH was monitored and maintained at 7.5+0.1 daily. After 30 days, thetoxoid was concentrated and washed into borate buffered saline (BBS)using a pressure filtration cell (Amicon), and sterilized by filtration.Conversion of the enterotoxin to enterotoxoid was confirmed by theabsence of weight loss in 3 to 3.5 kg rabbits injected intramuscularlywith 1 mg of toxoided material.

Immunizations

Microencapsulated and nonencapsulated antigens were suspended at anappropriate concentration in a solution of 8 parts filter sterilized tapwater and 2 parts sodium bicarbonate (7.5% solution). The recipient micewere fasted overnight prior to the administration of 0.5 mL ofsuspension via gastric intubation carried out with an intubation needle(Babb, J. L., Kiyono, H., Michalek, S. M. and McGhee, J. R. LPSregulation of the immune response: Suppression of immune response toorally-administered T-dependent antigen. J. Immunol. 127:1052; 1981).

Collection of Biological Fluids

1. Plasma

Blood was collected in calibrated capillary pipettes following punctureof the retro-orbital plexus. Following clot formation, the serum wascollected, centrifuged to remove red cells and platelets,heat-inactivated, and stored at -70° C. until assayed.

2. Intestinal Secretions

Mice were administered four doses (0.5 mL) of lavage solution 25 mMNaCl, 40 mM Na₂ SO₄, 10 mM KCl, 20 mM NaHCO₃, and 48.5 mM poly(ethyleneglycol), osmolarity of 530 mosM! at 15-minute intervals (Elson, C. O.,Ealding, W. and Lefkowitz, J. A lavage technique allowing repeatedmeasurement of IgA antibody on mouse intestinal secretions. J. Immunol.Meth. 67:101; 1984). Fifteen minutes after the last dose of lavagesolution, the mice were anesthetized and after an additional 15 minutesthey were administered 0.1 mg pilocarpine by ip injection. Over the next10 to 20 minutes, a discharge of intestinal contents was stimulated.This was collected into a petri dish containing 3 mL of a solution of0.1 mg/mL soybean trypsin inhibitor (Sigma, St. Louis, Mo.) in 50 mMEDTA, vortexed vigorously and centrifuged to remove suspended matter.The supernatant was transferred to a round-bottom, polycarbonatecentrifuge tube and 30 microliters of 20 millimolar phenylmethylsulfonylfluoride (PMSF, Sigma) was added prior to clarification by high-speedcentrifugation (27,000×g, 20 minutes, 4° C.). After clarification, 20microliters each of PMSF and 1% sodium azide were added and the solutionmade 10% in FCS to provide an alternate substrate for any remainingproteases.

3. Saliva

Concurrent with the intestinal discharge, a large volume of saliva issecreted and 0.25 mL was collected into a pasteur pipette by capillaryaction. Twenty microliters each of trypsin inhibitor, PMSF, sodium azideand FCS was added prior to clarification.

4. Bronchial-Alveolar Wash Fluids

Bronchial-alveolar wash fluids were obtained by lavaging the lungs with1.0 mL of PBS. An animal feeding needle was inserted intratracheally andfixed in place by tying with suture material. The PBS was inserted andwithdrawn 5 times to obtain washings, to which were added 20 microliterseach of trypsin inhibitor, PMSF, sodium azide, and FCS prior toclarification by centrifugation.

5. Immunochemical Reagents

Solid-phase absorbed and affinity-purified polyclonal goat IgGantibodies specific for murine IgM, IgG and IgA were obtainedcommercially (Southern Biotechnology Associates, Birmingham, Ala.).Their specificity in radioimmunoassays was tested through their abilityto bind appropriate purified monoclonal antibodies and myeloma proteins.

6. Solid-Phase Radioimmunoassays

Purified antibodies were labeled with carrier-free Na ¹²⁵ l (Amersham)using the chloramine T method Hunter, W. M. Radioimmunoassay. In:Handbook of Experimental Immunology, M. Weir (editor). BlackwellScientific Publishing, Oxford, p. 14.1; 1978). Immulon Removawell assaystrips (Dynatech) were coated with TNP conjugated bovine serum albumin(BSA) or staphylococcal enterotoxin B at 1 microgram/mL in BBS overnightat 4° C. Control strips were left uncoated but all strips were blockedfor 2 hours at room temperature with 1% BSA in BBS, which was used asthe diluent for all samples and ¹²⁵ l-labeled reagents. Samples ofbiologic fluids were appropriately diluted, added to washed triplicatereplicate wells, and incubated 6 hours at room temperature. Afterwashing, 100,000 cpm of ¹²⁵ l-labeled isotype-specificanti-immunoglobulin was added to each well and incubated overnight at 4°C. Following the removal of unbound ¹²⁵ l-antibodies by washing, thewells were counted in a Gamma 5500 spectrometer (Beckman Instruments,Inc., San Ramon, Calif.). In the case of the assays for TNP specificantibodies, calibrations were made using serial twofold dilutions of astandard serum (Miles Scientific, Naperville, Ill.) containing knownamounts of immunoglobulins, on wells coated with 1 microgram/wellisotype-specific antibodies. Calibration curves and interpolation ofunknowns was obtained by computer, using "Logit-log" or "Four ParameterLogistic" BASIC Technology Center (Vanderbilt Medical Center, Nashville,Tenn.). In the case of antibodies specific to staphylococcal enterotoxinB, the results are presented as the reciprocal serum dilution producinga signal >3-fold that of the group-matched prebleed at the same dilution(end-point titration).

A. Vaccine-Microcapsules Administered by Injection

1. Adjuvant Effect Imparted by Microencapsulation

EXAMPLE 1

Adjuvant Effect Imparted by Microencapsulation-IntraperitonealAdministration

Research in our laboratories has shown that microencapsulation resultsin a profoundly heightened immune response to the incorporated antigenor vaccine in numerous experimental systems. An example is provided bythe direct comparison of the level and isotype distribution of thecirculating antibody response to Staphylococcal enterotoxin B, thecausative agent of Staphylococcal food poisoning, following immunizationwith either soluble or microencapsulated enterotoxoid. Groups of micewere administered various doses of the toxoid vaccine incorporated in50:50 poly(DL-lactide-co-glycolide) microcapsules, or in soluble form,by intraperitoneal (IP) injection. On Days 10 and 20 followingimmunization, plasma samples were obtained and assayed for anti-toxinactivity by end-point titration in isotype-specific immunoradiometricassays (Table 4). The optimal dose of soluble toxoid (25 micrograms)elicited a characteristically poor immune response to the toxin whichwas detected only in the IgM isotype. In contrast, the administration of25 micrograms of toxoid incorporated within microcapsules induced notonly an IgM response, but an IgG response which was detectable at aplasma dilution of 1/2,560 on Day 20 post immunization. In addition,larger doses of toxoid could be administered in microencapsulated formwithout decreasing the magnitude of the response, as is seen with the 50microgram dose of soluble toxoid. In fact, the measured release achievedwith the microcapsules allows for 4-5 times the dose to be administeredwithout causing high zone paralysis, resulting in substantiallyheightened immunity. This adjuvant activity is even more pronouncedfollowing secondary (Table 5) and tertiary immunizations (Table 6).

The Day 20 IgG anti-toxin response following secondary immunization was512 times higher in mice receiving 50 micrograms of microencapsulatedtoxoid than in mice receiving the optimal dose of soluble toxoid.Further, tertiary immunization with the soluble toxoid at its optimaldose was required to raise an antibody response to the toxin which wasequivalent to that observed following a single immunization with 100micrograms of microencapsulated enterotoxoid. Adjuvant activity of equalmagnitude has been documented to common laboratory protein antigens suchas haptenated keyhole limpet hemocyanin and influenza virus vaccine.

EXAMPLE 2

Adjuvant Effect Imparted by Microencapsulation-SubcutaneousAdministration.

The present delivery system was found to be active followingintramuscular or subcutaneous (SC) injection. This was investigated bydirectly comparing the time course and level of the immune responsefollowing IP and SC injection into groups of mice, as shown in Table 7.

One hundred micrograms of enterotoxoid in microspheres administered bySC injection at 4 sites along the backs of mice stimulated a peak IgGanti-toxin response equivalent to that observed following IP injection.Some delay in the kinetics of anti-toxin appearance were observed.However, excellent antibody levels were attained, demonstrating theutility of injection at sites other than the peritoneum. Followingsecondary immunization the IP and SC routes were again equivalent withrespect to peak titer, although the delayed response of the SC route wasagain evident, as shown in Table 8.

2. Mechanism of the Adjuvant Effect Imparted by Microencapsulation.

EXAMPLE 1

The Adjuvant Effect Imparted by Microencapsulation is Not the Result ofAdjuvant Activity Intrinsic to the Polymer

When considering the mechanism through which 1-10 micrometer DL-PLGmicrospheres mediate a potentiated humoral immune response to theencapsulated antigen, three mechanisms must be considered aspossibilities. First, the long term chronic release (depot), as comparedto a bolus dose of nonencapsulated antigen, may play a role in immuneenhancement. Second, our experiments have shown that microspheres inthis size range are readily phagocytized by antigen processing andpresenting cells. Therefore, targeted delivery of a comparatively largedose of nondegraded antigen directly to the cells responsible for theinitiation of immune responses to T cell-dependent antigens must also beconsidered. Third, the microcapsules may possess intrinsicimmunopotentiating activity through their ability to activate cells ofthe immune system in a manner analogous to adjuvants such as bacteriallipopolysaccharide or muramyl-di-peptide. Immunopotentiation by thislatter mechanism has the characteristic that it is expressed when theadjuvant is administered concurrently with the antigen.

In order to test whether microspheres possess any innate adjuvancy whichis mediated through the ability of these particles to nonspecificallyactivate the immune system, the antibody response to 100 micrograms ofmicroencapsulated enterotoxoid was compared to that induced followingthe administration of an equal dose of enterotoxoid mixed with placebomicrospheres containing no antigen. The various antigen forms wereadministered by IP injections into groups of 10 BALB/c mice and theplasma IgM and IgG enterotoxin-specific antibody responses determined byend-point titration RIAs, as shown in Table 9.

The plasma antibody response to a bolus injection of the optimal dose ofsoluble enterotoxoid (25 micrograms) was characteristically poor andconsisted of a peak IgM titer of 800 on day 10 and a peak IgG titer of800 on day 20. Administration of an equal dose of microencapsulatedenterotoxoid induced a strong response in both the IgM and IgG isotypeswhich was still increasing on day 30 after immunization.Co-administration of soluble enterotoxoid and a dose of placebomicrospheres equal in weight, size and composition to those used toadminister encapsulated antigen did not induce a plasma anti-toxinresponse which was significantly higher than that induced by solubleantigen alone. This result was not changed by the administration of thesoluble antigen 1 day before or 1, 2 or 5 days after the placebomicrospheres. Thus, these data indicate that the immunopotentiationexpressed when antigen is administered within 1-10 micrometer DL-PLGmicrospheres is not a function of the ability of the microspheres tointrinsically activate the immune system. Rather, the data areconsistent with either a depot effect, targeted delivery of the antigento antigen-presenting accessory cells, or a combination of these twomechanisms.

EXAMPLE 2

Retarding the Antigen-Release Rate from 1-10 Micrometer MicrocapsulesIncreases the Level of the Antibody Response and Delays the the Time ofthe Peak Response

Four enterotoxoid containing microcapsule preparations with a variety ofantigen release rates were compared for their ability to induce a plasmaanti-toxin response following IP injection. The rate of antigen releaseby the microcapsules used in this study is a function of two mechanisms;diffusion through pores in the wall matrix and hydrolysis (bioerosion)of the wall matrix. Batches #605-026-1 and #514-140-1 have varyinginitial rates of release through pores, followed by a second stage ofrelease which is a function of their degradation through hydrolysis. Incontrast, Batches #697-143-2 and #928-060-00 have been manufactured witha tight uniform matrix of wall material which has little release throughpores and their release is essentially a function of the rate at whichthe wall materials are hydrolyzed. However, these latter two lots differin the ratio of lactide to glycolide composing the microcapsules, andthe greater resistance of the 85:15 DL-PLG to hydrolysis results in aslower rate of enterotoxoid release.

The immune response induced by Batch #605-026-1 (60% release at 48hours) reached a peak IgG titer of 6,400 on day 20 (Table 10). Batch#514-140-1 (30% release at 48 hours) stimulated IgG antibodies whichalso peaked on day 20, but which were present in higher concentrationboth on days 20 and 30.

Immunization with Batch #697-143-2 (10% release at 48 hours) resulted inpeak IgG antibody levels on days 30 and 45 which were substantiallyhigher (102,400) than those induced by either lot with early release.Further delaying the rate of antigen release through the use of an 85:15ratio of lactide to glycolide, Batch #928-060-00 (0% release at 48hours) delayed the peak antibody levels until days 45 and 60, but nofurther increase in immunopotentiation was observed.

These results are consistent with a delayed and sustained release ofantigen stimulating a higher antibody response. However, certain aspectsof the pattern of responses induced by these various microspheresindicate that a depot effect is not the only mechanism ofimmunopotentiation. The faster the initial release, the lower the peakantibody titer. These results are consistent with a model in which theantigen released within the first 48 hours via diffusion through poresis no more effective than the administration of soluble antigen.Significant delay in the onset of release to allow time for phagocytosisof the microspheres by macrophages allows for the effective processingand presentation of the antigen, and the height of the resultingresponse is governed by the amount of antigen delivered into thepresenting cells. However, delay of antigen release beyond the pointwhere all the antigen is delivered into the presenting cells does notresult in further potentiation of the response, it only delays the peaklevel.

EXAMPLE 3

Correlation of the Size of the Microcapsules with the Resultant AdjuvantEffect

It has been consistently observed that the size of the microspheres hasa profound effect on the degree to which the antibody response ispotentiated and the time at which it is initiated. These effects arebest illustrated under conditions of a limiting antigen dose. Miceimmunized subcutaneously with 10 μg of SEB toxoid encapsulated in 1-10μm microspheres produced a more rapid, and a substantially morevigorous, IgG anti-toxin response than did mice immunized with the samedose of toxoid in 10-110 μm microspheres as shown in FIG. 1. Groups of 5mice were subcutaneously immunized with 10 μg of SEB toxoid encapsulatedin 1-10 μm (85:15 DL-PLG; 0.065 wt % SEB toxoid) or 10-110 μm (85:15DL-PLG; 1.03 wt % SEB toxoid) microspheres. Plasma samples were obtainedat 10 day intervals and the IgG anti-toxin titer determined by end-pointtitration in a RIA using solid-phase adsorbed SEB toxin.

A likely explanation for these effects involves the manner in whichthese different sizes of microspheres deliver antigen into the draininglymphatics. We have observed fluorescent DL-PLG microspheres of <10 μmin diameter to be efficiently phagocytized and transported bymacrophages into the draining lymph nodes. In contrast, largermicrospheres (>10 μm)remain localized at the site of injection. Takentogether, these data suggest that the extremely strong adjuvant activityof <10 μm microspheres is due to their efficient loading of antigen intoaccessory cells which direct the delivery of the microencapsulatedantigen into the draining lymph nodes.

3. Pulsatile Release of Vaccines from Microcapsules for ProgrammedBoosting Following a Single Injection

When one receives any of a number of vaccines by injection, two to threeor more administrations of the vaccine are required to illicit a goodimmune response. Typically, the first injection is given to afford aprimary response, the second injection is given to afford a secondaryresponse, and a third injection is given to afford a tertiary response.Multiple injections are needed because repeated interaction of theantigen with immune system cells is required to stimulate a strongimmunological response. After receiving the first injection of vaccine,a patient, therefore, must return to the physician on several occasionsto receive the second, third, and subsequent injections to acquireprotection. Often patients never return to the physician to get thesubsequent injections.

The vaccine formulation that is injected into a patient may consist ofan antigen in association with an adjuvant. For instance, an antigen canbe bound to alum. During the first injection, the use of theantigen/adjuvant combination is important in that the adjuvant aids inthe stimulation of an immune response. During the second and thirdinjections, the administration of the antigen improves the immuneresponse of the body to the antigen. The second and thirdadministrations or subsequent administrations, however, do notnecessarily require an adjuvant.

Alza Corporation has described methods for the continuous release of anantigen and an immunopotentiator (adjuvant) to stimulate an immuneresponse (U.S. Pat. No. 4,455,142). This invention differs from the Alzapatent in at least two important manners. First, no immunopotentiator isrequired to increase the immune response, and second, the antigen is notcontinuously released from the delivery system.

The present invention concerns the formulation of vaccine (antigen) intomicrocapsules (or microspheres) whereby the antigen is encapsulated inbiodegradable polymers, such as poly(DL-lactide-co-glycolide). Morespecifically, different vaccine microcapsules are fabricated and thenmixed together such that a single injection of the vaccine capsulemixture improves the primary immune response and then delivers antigenin a pulsatile fashion at later time points to afford secondary,tertiary, and subsequent responses.

The mixture of microcapsules may consist of small and largemicrocapsules. The small microcapsules, less than 10 microns, preferablyless than 5 micrometers, or more preferable 1 to 5 micrometers,potentiate the primary response (without the need of an adjuvant)because the small microcapsules are efficiently recognized and taken upby macrophages. The microcapsules inside of the macrophages then releasethe antigen which is subsequently processed and presented on the surfaceof the macrophage to give the primary response. The largermicrocapsules, greater than 5 micrometers, preferably greater than 10microns, but not so large that they cannot be administered for instanceby injection, preferably less than 250 micrometers, are made withdifferent polymers so that as they biodegrade at different rates, theyrelease antigen in a pulsatile fashion.

Furthermore, the mixture of microcapsules may consist entirely ofmicrocapsules sized less than 10 micrometers. Microspheres less than 10micrometers in diameter are rapidly phagocytized by macrophages afteradministration. By using mixtures of microspheres less than 10micrometers in diameter that have been prepared with polymers that havevarious lactide/glycolide ratios, an immediate primary immunization aswell as one or more discrete booster immunizations at the desiredintervals (up to approximately eight months after administration) can beobtained. By mixing microspheres less than 10 micrometers in diameter(for the primary immunization) with microspheres greater than 10micrometers in diameter, the time course possible for delivery of thediscrete booster immunizations can be extended up to approximately 2years. This longer time course is possible because the largermicrospheres are not phagocytized and are therefore degraded at a slowerrate than are the less than 10 micrometer microspheres.

Using the present invention, the composition of the antigenmicrocapsules for the primary response is basically the same as thecomposition of the antigen microcapsules used for the secondary,tertiary, and subsequent responses. That is, the antigen is encapsulatedwith the same class of biodegradable polymers. The size and pulsatilerelease properties of the antigen microcapsules then maximizes theimmune response to the antigen.

The preferred biodegradable polymers are those whose biodegradationrates can be varied merely by altering their monomer ratio, for example,poly(DL-lactide-co-glycolide), so that antigen microcapsules used forthe primary response will biodegrade faster than antigen microcapsulesused for subsequent responses, affording pulsatile release of theantigen.

In summary, by controlling the size of the microcapsules of basicallythe same composition, one can maximize the immune response to anantigen. Also important is having small microcapsules (microcapsulesless than 10 micrometers, preferably less than 5 micrometers, mostpreferably 1 to 5 micrometers) in the mixture of antigen microcapsulesto maximize the primary response. The use of an immune enhancingdelivery system, such as small microcapsules, becomes even moreimportant when one attempts to illicit an immune response to lessimmunogenic compounds such as killed vaccines, subunit vaccines,low-molecular-weight vaccines such as peptides, and the like.

EXAMPLE 1

Co-administration of Free and Microencapsulated Vaccine.

A Japanese Encephalitis virus vaccine (Biken) was studied. The virusused is a product of the Research Foundation for Microbial Disease ofOsaka University, Suita, Osaka, Japan. The manufacturer recommends athree dose immunization series consisting of two doses of vaccineadministered one to two weeks apart followed by administration of athird dose of vaccine one month after the initial immunization series.We have compared the antiviral immune responses of mice immunized with astandard three dose schedule of JE vaccine to the antiviral response ofmice immunized with a single administration of JE vaccine consisting ofone part unencapsulated vaccine and two parts encapsulated vaccine. TheJE microcapsules were >10 micrometers. The results of immunizing micewith JE vaccine by these two methods were compared by measuring theserum antibody titers against JE vaccine detected through an ELISAassay. The ELISA assay measures the presence of serum antibodies withspecificity of JE vaccine components, however, it does not measure thelevel of virus neutralizing antibody present in the serum. The virusneutralizing antibody activity was therefore measured by viruscytopathic effect (CPE) inhibition assays and virus plaque reductionassays. The results of those assays are presented here.

Four experimental groups consisting of (1) untreated control mice whichreceive no immunization; (2) mice which received 3.0 mg of JE vaccine(unencapsulated) on Day 0; (3) mice which received 3.0 mg of JE vaccine(unencapsulated) on Days 0, 14 and 42 (standard schedule) and (4) micewhich received 3.0 mg of JE vaccine (unencapsulated) and 3.0 mg of JEvaccine (encapsulated) on day 0 were studied. The untreated controlsprovide background virus neutralization titers against which immunizedanimals can be compared. The animals receiving a single 3.0 mg dose ofJE vaccine on Day 0 provide background neutralization titers againstwhich animals receiving unencapsulated vaccine in conjunction withencapsulated vaccine can be compared. This comparison provides evidencethat the administration of encapsulated vaccine augments theimmunization potential of a single 3.0 mg dose of unencapsulatedvaccine. The animals receiving 3 doses of unencapsulated vaccine providecontrols against which the encapsulated vaccine group can be compared soas to document the ability of a single injection consisting of bothnonencapsulated and encapsulated vaccine to produce antiviral activitycomparable to a standard three dose immunization schedule.

Serum samples collected on Days 21, 49 and 77 from ten animals in eachexperimental group were tested for their ability to inhibit thecytopathic effects induced by a standard challenge (100 TCID₅₀) of JEvirus. The results of the CPE inhibition assays, expressed as thehighest serum dilution capable of inhibiting 50% of the viral CPE, arepresented in Table 11. As is shown, the untreated control animals(Group 1) had no significant serum virus neutralizing activity at anypoint tested. Of the ten animals receiving a single 3.0 mg dose of JEvaccine on Day 0 (Group 2), one did not develop any detectable virusneutralizing antibody. Of the remaining nine mice, the highest titerachieved was 254 which occurred on Day 49. The geometric mean antiviraltiter for this experimental group peaked on Day 49. Of the ten animalsreceiving a standard schedule of three vaccine doses (Group 3), eighthad a decrease in antibody activity from Day 49 to Day 77. The geometricmean titer for this group decreased by greater than 50% from Day 40 toDay 77. All ten animals receiving encapsulated JE vaccine (Group 4)developed serum antiviral activity. The geometric mean titer for thisgroup increased from Day 21 to Day 77. The average titer occurring onDay 49 in this group was significantly lower than that occurring in the3 vaccine dose group (Group 3) (p=0.006); however, the titer continuedto increase from Day 49 to Day 77 which is in contrast to the 3 vaccinedose group. There was no significant difference in the average titer forthese two groups in the Day 77 samples (p=0.75) indicating that theencapsulated vaccine group achieved comparable serum antiviral titers atDay 77. Unlike the 3 vaccine dose group (Group 3), the animals receivingencapsulated vaccine (Group 4) continued to demonstrate increases inserum virus neutralizing activity throughout the timepoints examined. Incontrast to the standard vaccine treatment group, mice receivingencapsulated JE vaccine had a two-fold increase in the average serumneutralizing titer from Day 49 to Day 77. The Day 21 average antiviraltiter from mice receiving microencapsulated vaccine was notsignificantly different from the Day 21 average titer of mice receivinga single dose of JE vaccine on Day 0 (p=0.12); however, the day 49 andDay 77 average titers were significantly different for the two groups(p=0.03 and p=0.03, respectively). These data indicate that serum virusneutralizing titers similar to those produced by standard vaccineadministration can be achieved by administering a single dose ofencapsulated JE vaccine. Although the antiviral titers achieved with theexcipient formulation used in this study did not increase as rapidly asthose achieved with the standard vaccine, the serum neutralizingantibody activity did reach titers which are comparable to thoseachieved with the standard three dose vaccine schedule.

To further corroborate these findings, pooled samples produced by mixingequal volumes of each serum sample were prepared for each experimentalgroup. These samples were submitted to an independent laboratory fordetermination of antiviral activity. The samples were tested by plaquereduction assay against a standard challenge of JE virus. The results ofthese assays, presented in Table 12, substantiate the findings describedabove. Although the animals receiving encapsulated vaccine did not reachpeak titers as rapidly as did the standard vaccine group, theencapsulated vaccine did induce comparable virus neutralizing antibodyactivity. Furthermore, the encapsulated vaccine maintained a higherantiviral titer over a longer period of time than did the standardvaccine. These results further support the conclusion that a singleadministration of microencapsulated vaccine can produce resultscomparable to those achieved with a three dose schedule of standardvaccine.

EXAMPLE 2

Co-administration of <10 Micrometer Priming and >10 Micrometer PulsingVaccine Microcapsules

One advantage of the copolymer microcapsule delivery system is theability to control the time and/or rate at which the incorporatedmaterial is released. In the case of vaccines this allows for schedulingof the antigen release in such a manner as to maximize the antibodyresponse following a single administration. Among the possible releaseprofiles which would be expected to improve the antibody response to avaccine is a pulsed release (analogous to conventional boosterimmunizations).

The possibility of using size as a mechanism to control vaccine releaseis based on the observation that microspheres <10 micrometers indiameter are phagocytized by macrophages and release antigen at asubstantially accelerated rate relative to microspheres made of the sameDL-PLG but which are too large to be phagocytized. The possibility ofusing size to achieve pulsed vaccine release was investigated bysystemically (subcutaneously) injecting 100 micrograms of enterotoxoidto groups of mice either in 1-10 micrometer (50:50 DL-PLG; 1.51 wt %enterotoxoid), 20-50 micrometers (50:50 DL-PLG; 0.64 wt % enterotoxoid)or in a mixture of 1-10 micrometer and 20-50 micrometer microcapsules inwhich equal parts of the enterotoxoid were contained within each sizerange. The groups of mice were bled at 10 day intervals and the plasmaIgG responses were determined by endpoint titration in isotype-specificimmunoradiometric assays employing solid-phase absorbed enterotoxin(FIG. 2). Following the administration of the 1-10 micrometerenterotoxoid microcapsules the plasma IgG response was detected on day10, rose to a maximal titer of 102,400 on days 30 and 40, and decreasedthrough day 60 to 25,600. In contrast, the response to the toxoidadministered in 20-50 micrometer microcapsules was delayed until day 30,and thereafter increased to a titer of 51,200 on days 50 and 60. Theconcomitant administration of equal parts of the toxoid in 1-10 and20-50 micrometer microcapsules produced an IgG response which was forthe first 30 days essentially the same as that stimulated by the 1-10micrometer microcapsules administered alone. However, beginning on day40 the response measured in the mice concurrently receiving the 1-10plus 20-50 micrometer microcapsules steadily increased to a titer of819,200 on day 60, a level which was far more than the additive amountof the responses induced by the two size ranges administered singly.

The antibody response obtained through the co-administration of 1-10 and20-50 micrometer enterotoxoid-containing microcapsules is consistentwith a two phase (pulsed) release of the antigen. The first pulseresults from the rapid ingestion and accelerated degradation of the 1-10micrometer particles by tissue histiocytes, which results in apotentiated primary immune response due to the efficient loading of highconcentrations of the antigen into these accessory cells, and mostprobably their activation. The second phase of antigen release is due tothe biodegradation of the 20-50 micrometer microcapsules, which are toolarge to be ingested by phagocytic cells. This second pulse of antigenis released into a primed host and stimulates an anamnestic immuneresponse. Thus, using the 50:50 DL-PLG copolymer, a single injectionvaccine delivery system can be constructed which potentiates antibodyresponses (1-10 micrometer microcapsules), and which can deliver a timedand long lasting secondary booster immunization (20-50 micrometermicrocapsules). In addition, through alteration of the ratio of thecopolymers, it is possible to prepare formulations which release evenlater, in order to provide tertiary or even quaternary boostings withoutthe need for additional injections.

EXAMPLE 3

Co-administration of <10 Micrometer Priming and <10 Micrometer PulsingVaccine Microcapsules

The hydrolysis rate of the DL-PLG copolymer can be changed by alteringthe lactide-to-glycolide ratio. This approach to the pulsed release ofvaccine antigens was investigated in experiments in which groups of micewere subcutaneously immunized with 10 μg of SEB toxoid in 1 to 10micrometer microspheres formulated from DL-PLG with lactide-to-glycolideratios of 50:50 or 85:15 DL-PLG or 100:0 L-PLG. Determination of theplasma IgG anti-toxin levels in these mice as a function of timedemonstrated that these preparations of microencapsulated SEB toxoidstimulated an anti-SEB toxin response which both initiated and peaked atdistinctly different times as shown in FIG. 3. Each preparationstimulated a peak IgG titer of 409,600, but the microspheres formulatedof 50:50 and 85:15 DL-PLG and 100:0 L-PLG resulted in this level beingattained on days 50, 130 and 230, respectively.

The possibility of using a blend of 1 to 10 μm microspheres withdifferent DL-PLGs having different lactide/glycolide ratios to deliverdiscrete pulsed releases of antigen was investigated in a group of micesubcutaneously immunized in parallel. This blend consisted of 50:50DL-PLG and 100:0 L-PLG microspheres in which each component contained 5μg of SEB toxoid. The plasma IgG anti-SEB toxin response induced by thismixture was distinctly biphasic and exhibited both primary and secondarycomponents as shown in FIG. 4. The first component of this response wascoincident with that seen in mice which received only the 50:50 DL-PLGmicrospheres, while the second component coincided with the time atwhich the immune response was observed in mice receiving only the 100:0L-PLG microspheres. The anamnestic character of the second phaseindicates that distinct primary and secondary anti-SEB toxin responseshave been induced.

These data show-that in a mixture of microspheres with differinglactide/glycolide ratios, the degradation rate of an individualmicrosphere is a function of its lactide/glycolide ratio and that it isindependent of the degradation rate of the other microspheres in themixture. This finding indicates that 1) the time at which any vaccinepulse can be delivered is continuously variable across the range oflactide/glycolide rations, 2) the pulsed vaccine release profiles of anycombination of microspheres with differing lactide/glycolide ratios canbe predicted with a high degree of certainty based on the behavior ofthe individual components, and 3) the delay in vaccine release possiblewith microspheres <10 μm in diameter is up to approximately 8 monthswhile the delay possible for microspheres >10 μm is up to approximately2 years, allowing for any number of discrete pulsatile vaccine releasesover these time frames.

Therefore, there exist a number of possible approaches to vaccination bythe injectable microcapsules of the present invention. Among theseinclude multiple injections of small microcapsules, preferably 1 to 5micrometers, that will be engulfed by macrophages and obviate the needfor immunopotentiators, as well as mixtures of free antigen for aprimary response in combination with microcapsulated antigen in the formof microcapsules having a diameter of 10 micrometers or greater thatrelease the antigen pulsatile to potentiate secondary and tertiaryresponses and provide immunization with a single administration. Also, acombination of small microcapsules for a primary response and largermicrocapsules for secondary and later responses may be used, therebyobviating the need for both immunopotentiators and multiple injections.

B. Vaccine-Microcapsules Administered Orally

EXAMPLE 1

Orally-Administered Microspheres Containing TNP-KLH Induce ConcurrentCirculating and Mucosal Antibody Responses to TNP

Microcapsules containing the haptenated protein antigentrinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) were prepared using50:50 DL-PLG as the excipient. These microcapsules were separatedaccording to size and those in the range of 1 to 5 micrometers indiameter were selected for evaluation. These microcapsules contained0.2% antigen by weight. Their ability to serve as an effective antigendelivery system when ingested was tested by administering 0.5 mL of a 10mg/mL suspension (10 micrograms antigen) in bicarbonate-buffered steriletap water via gastric incubation on 4 consecutive days. For comparativepurposes an additional group of mice was orally immunized in parallelwith 0.5 mL of 20 micrograms/mL solution of unencapsulated TNP-KLH.Control mice were orally administered diluent only.

On Days 14 and 28 following the final immunization, serum, saliva andgut secretions were obtained from 5 fasted mice in each group. Thesesamples were tested in isotype-specific radioimmunoassays to determinethe levels of TNP-specific and total antibodies of the IgM, IgG and IgAisotypes (Table 13). The samples of saliva and gut secretions containedantibodies which were almost exclusively of the IgA class. These resultsare consistent with previous studies and provide evidence that theprocedures employed to collect these secretions do not result incontamination with serum. None of the immunization protocols resulted insignificant changes in the total levels of immunoglobulins present inany of the fluids tested. Low but detectable levels ofnaturally-occurring anti-TNP antibodies of the IgM and IgG isotypes weredetected in the serum and of the IgA isotype in the serum and gutsecretions of sham immunized control mice. However, the administrationof 30 micrograms of microencapsulated TNP-KLH in equal doses over 3consecutive days resulted in the appearance of significantantigen-specific IgA antibodies in the secretions, and of all isotypesin the serum by Day 14 after immunization (see the last column of Table13). These antibody levels were increased further on Day 28. Incontrast, the oral administration of the same amount of unencapsulatedantigen was ineffective at inducing specific antibodies of any isotypein any of the fluids tested.

These results are noteworthy in several respects. First, significantantigen-specific IgA antibodies are induced in the serum and mucosalsecretions, a response which is poor or absent following the commonlyused systemic immunization methods. Therefore, this immunization methodwould be expected to result in significantly enhanced immunity at themucosa; the portal of entry or site of pathology for a number ofbacterial and viral pathogens. Secondly, the microencapsulated antigenpreparation was an effective immunogen when orally administered, whilethe same amount of unencapsulated antigen was not. Thus, themicroencapsulation resulted in a dramatic increase in efficacy, due totargeting of and increased uptake by the Peyer's patches. Thirdly, theinductive phase of the immune response appears to be of long duration.While systemic immunization with protein antigens in the absence ofadjuvants is characterized by a peak in antibody levels in 7 to 14 days,the orally administered antigen-containing microcapsules inducedresponses were higher at Day 28 than Day 14. This indicates thatbioerosion of the wall materials and release of the antigen is takingplace over an extended period of time, and thus inducing a response ofgreater duration.

EXAMPLE 2

Orally Administered Microcapsules Containing SEB Toxoid InduceConcurrent Circulating and Mucosal Anti-SEB Toxin Antibodies

The results presented above which show that (a) strong adjuvant activityis imparted by microencapsulation, and (b) microcapsules <5 micrometersin diameter disseminate to the mesenteric lymph nodes and spleen afterentering through the Payer's patches, suggested that it would befeasible to induce a systemic immune response by oral immunization withvaccine incorporated into appropriately sized biodegradablemicrocapsules. This possibility was confirmed in experiments in whichgroups of mice were immunized with 100 micrograms of Staphylococcalenterotoxoid B in soluble form or within microcapsules with a 50:50DL-PLG excipient. These mice were administered the soluble ormicroencapsulated toxoid via gastric tube on three occasions separatedby 30 days, and plasma samples were obtained on Days 10 and 20 followingeach immunization. The data presented in Table 14 show the plasma endpoint titers of the IgM and IgG anti-toxin responses for the Day 20 timepoint after the primary, secondary and tertiary oral immunizations.

Mice receiving the vaccine incorporated in microcapsules exhibited asteady rise in plasma antibodies specific to the toxin with eachimmunization while soluble enterotoxoid was ineffective. This experimentemployed the same lot of microcapsules and was performed and assayed inparallel with the experiments presented in Tables 4, 5 and 6 above.Therefore, these data directly demonstrate that oral immunization withmicroencapsulated Staphylococcal enterotoxoid B is more effective atinducing a serum anti-toxin response than is the parenteral injection ofthe soluble enterotoxoid at its optimal dose.

The secretory IgA response was examined in the same groups of mice. Itwas reasoned that the characteristics of this lot ofenterotoxoid-containing microcapsules, a heterogeneous size range from<1 micrometer to approximately 10 micrometers, made it likely that aproportion of the microcapsules released the toxoid while fixed in thePeyer's patches. Therefore, on Days 10 and 20 following the tertiaryoral immunization saliva and gut wash samples were obtained and assayedfor toxin-specific antibodies of the IgA isotype (Table 15). In contrastto the inability of the soluble toxoid to evoke a response whenadministered orally, the ingestion of an equal amount of the toxoidvaccine incorporated into microcapsules resulted in a substantial sIgAanti-toxoid response in both the saliva and gut secretions. It should bepointed out that the gut secretions from each mouse are diluted into atotal of 5 mL during collection. Although it is difficult to determinethe exact dilution factor this imposes on the material collected, it issafe to assume that the sIgA concentration is at minimum 10-fold higherin the mucus which bathes the gut, and this has not been taken intoaccount in the measurements present here.

These data clearly demonstrate the efficacy of microencapsulatedenterotoxoid in the induction of a sIgA anti-toxin response in both thegut and at a distant mucosal site when administered orally. Furthermore,through the use of a mixture of microcapsules with a range of diametersfrom <1 to 10 micrometers it is possible to induce this mucosal responseconcomitant with a strong circulating antibody response. This suggeststhat a variety of vaccines can be made both more effective andconvenient to administer through the use of microencapsulationtechnology.

C. Vaccine Microcapsules Administered Intratracheally

EXAMPLE 1

Intratracheally Administered Microcapsules Containing SEB Toxoid InduceConcurrent Circulating and Mucosal Anti-Toxin Antibodies

Folliculi lymphatic aggregati similar to the Peyer's patches of thegastrointestinal tract are present in the mucosally-associated lymphoidtissues found at other anatomical locations, such as the respiratorytract. Their function is similar to that of the Peyer's patches in thatthey absorb materials from the lumen of the lungs and are inductivesites for antibody responses which are characterized by a highproportion of sIgA. The feasibility of immunization through thebronchial-associated lymphoid tissue was investigated. Groups of micewere administered 50 microliters of PBS containing 50 micrograms of SEBtoxoid in either microencapsulated or nonencapsulated form directly intothe trachea. On days 10, 20, 30 and 40 following the immunization,samples of plasma, saliva, gut washings and bronchial-alveolar washingswere collected.

Assay of the plasma samples for anti-toxin specific antibodies revealedthat the administration of free SEB toxoid did not result in theinduction of a detectable antibody response in any isotype (Table 16).In contrast, intratracheal instillation of an equal dose ofmicroencapsulated SEB vaccine elicited toxin specific antibodies of allisotypes. This response reached maximal levels on Day 30 and wasmaintained through day 40 with IgM, IgG and IgA titers of 400, 51,300and 400, respectively.

Similar to the responses observed in the plasma, toxin-specificantibodies in the bronchial-alveolar washings were induced by themicroencapsulated toxoid, but not by the nonencapsulated vaccine (Table17). The kinetics of the appearance of the anti-toxin antibodies in thebronchial secretions was delayed somewhat as compared to the plasmaresponse in that the Day 20 response was only detected in the IgGisotype and was low in comparison to the plateau levels eventuallyobtained. However, maximal titers of IgG and IgA anti-toxin antibodies(1,280 and 320, respectively) were attained by Day 30 and weremaintained through Day 40. No IgM class antibodies were detected in thebronchial-alveolar washings using this immunization method, a resultconsistent with the absence of IgM secreting plasma cells in the lungsand the inability of this large antibody molecule to transudate from theserum past the approximately 200,000 molecular weight cut off imposed bythe capillary-alveolar membrane.

These data demonstrate that microencapsulation allowed an immuneresponse to take place against the antigen SEB toxoid followingadministration into the respiratory tract while the nonencapsulatedantigen was ineffective. This response was,observed both in thecirculation and in the secretions bathing the respiratory tract. Itshould be noted that this immunization method was effective at inducingthe appearance of IgA class antibodies. This antibody is presumably theproduct of local synthesis in the upper respiratory tract, an area whichis not protected by the IgG class antibodies which enter the lower lungsfrom the blood circulation. Thus, intratracheal immunization withmicroencapsulated antigens, through the inhalation of aerosols, will bean effective means of inducing antibodies which protect the upperrespiratory tract.

D. Vaccine Microcapsules Administered by Mixed Immunization Routes

In both man and animals, it has been shown that systemic immunizationcoupled with mucosal presentation of antigen is more effective than anyother combination in promoting mucosal immune responses (Pierce, N. F.and Gowans, J. L. Cellular kinetics of the intestinal immune response tocholera toxoid in rats. J. Exp. Med. 142:1550; 1975). Three groups ofmice were primed by IP immunization with 100 micrograms ofmicroencapsulated SEB toxoid and 30 days later were challenged with 100micrograms of microencapsulated SEB toxoid by either the IP, oral or ITroutes. This was done to directly determine if a mixed immunizationprotocol utilizing microencapsulated antigen was advantageous withrespect to the levels of sIgA induced.

Twenty days following the microencapsulated booster immunizations,samples of plasma, gut washings and bronchial-alveolar washings wereobtained and the levels and isotype distribution of the anti-SEB toxinantibodies were determined in endpoint titration radioimmunoassays(Table 18). The IP boosting of IP primed mice led to the appearance ofhigh levels of IgG anti-toxin antibodies in the samples of plasma andsecretions, but was completely ineffective at the induction ofdetectable IgA antibodies in any fluid tested. In contrast, secondaryimmunization with microencapsulated SEB toxoid by either the oral or ITroutes efficiently boosted the levels of specific IgG antibodies in theplasma (pre-secondary immunization titer in each group was 51,200) andalso induced the appearance of significant levels of sIgA antibodies inthe gut and bronchial-alveolar washings. Oral boosting of IP primed miceinduced sIgA anti-SEB toxin antibodies to be secreted into the gutsecretions at levels which were comparable with those requiring threespaced oral immunizations (Table 18 as compared to Table 15).Intratracheal boosting of previously IP immunized mice was particularlyeffective in the induction of a disseminated mucosal response andelicited the appearance of high concurrent levels of IgG and sIgAantibodies in both the samples of bronchial-alveolar and gut secretions.

These results are particularly important with respect to immunizationagainst numerous infectious agents which exert their pathophysiologiceffects through acute infections localized to the respiratory tract.Antibodies present within the respiratory tract originate from twodifferent sources. Secretory IgA predominates in the mucus which bathesthe nasopharynx and bronchial tree (Soutar, C. A. Distribution of plasmacells and other cells containing immunoglobulin in the respiratory tractof normal man and class of immunoglobulin contained therein. Thorax31:58; 1976 and Kaltreider, H. B. and Chan, M. K. L. The class-specifiedimmunoglobulin composition of fluids obtained from various levels ofcanine respiratory tract. J. Immunol. 116:423; 1976) and is the productof local plasma cells which are located in the lamina propria of theupper respiratory tract. In contrast to the nasopharynx and bronchialtree, the bronchioli and alveoli predominantly contain IgG which ispassively-derived from the blood circulation via transudation (Reynolds,H. Y. and Newball, H. H. Analysis of proteins and respiratory cellsobtained from human lungs by bronchial lavage. J. Lab. Clin. Med.84:559; 1974). Thus, effective protection of the lungs requires bothcirculating IgG and mucosal sIgA antibodies.

These data indicate that mixed route immunization protocols utilizingmicroencapsulated antigens will prove the most efficient in theinduction of concurrent circulating and mucosal antibody responses.Although the experiments reported here examine discrete priming andboosting steps which each required an administration ofmicroencapsulated antigen, it will be possible to use the flexibility incontrolled pulsatile release afforded by the microcapsule deliverysystem to design a single time of administration regimen which willstimulate maximum concurrent systemic and secretory immunity. As anexample, microencapsulated antigen could be administered by bothinjection and ingestion during a single visit to a physician. By varyingthe lactide to glycolide ratio in the two doses, the systemicallyadministered dose could be released within a few days to prime theimmune system, and the second (oral) dose could be released in thePeyer's patches at a later time to stimulate a boosted mucosal response.

IV. ABSORPTION OF PHARMACEUTICALS

The following example shows that small microcapsules (less than 5micrometers, preferably 1 to 5 microns) can also improve the absorptionof pharmaceuticals as well as antigens into the body. Etretinate,(Al1-E)-9-(4-methoxy-2,3,6,-trimethyl) phenyl-3,7-dimethyl-2,4,8-nonatetraenoic acid, ethyl ester) was microencapsulatedin 50:50 poly(DL-lactide-co-glycolide). The microcapsules were 0.5 to 4micrometers in diameter and contained 37.2 wt % etretinate. Theseetretinate microcapsules, as well as unencapsulated etretinate, wasadministered to mice by oral gavage using 1 wt % Tween 80 in water as avehicle. Only single doses of 50 mg etretinate/kg were given. Blood fromthe dosed mice was collected at specific time intervals and the serum ofthis blood was quantified for etretinate and/or its metabolites using ahigh performance chromatographic procedure (Table 19). The results showthat mice treated with the,etretinate microcapsules had significantlyhigher blood levels of etretinate than mice treated with unencapsulatedetretinate. Like the less than 5-micrometer vaccine microcapsules, it isbelieved that the microcapsules carry the etretinate to the blood streamvia the lymphoidal tissue (Peyer's patches) in the gastrointestinaltract. This same approach should be applicable to increasing theabsorption of other drugs, where its application would be especiallyuseful for the delivery of biological pharmaceuticals such as peptides,proteins, nucleic acids, and the like.

                  TABLE 1                                                         ______________________________________                                        Penetration of Coumarin-6 85:15 DL-PLG Microspheres Into and                  Through the Peyer's Patches Following Oral Administration                     Total       Proportion of diameter (%)                                                                     Proportion at                                    Time  number    Small   Medium Large location (%)                             (days)                                                                              observed  <2 um   2-5 um >5 um Dome   Deep                              ______________________________________                                         1    296       47      35     18    92     8                                  2    325       45      32     23    83     17                                 4    352       46      31     23    76     24                                 7    196       21      29     41    88     11                                14    148       16      29     55    98     2                                 21     91        7      27     66    98     2                                 28     63        5      24     71    100    0                                 35     52        6      19     79    97     3                                 ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Migration of Coumarin-6 85:15 DL-PLG Microspheres Into and                    Through the Mesenteric Lymph Nodes Following Oral Administration              Total       Proportion of diameter (%)                                                                     Proportion at                                    Time  number    Small   Medium Large location (%)                             (days)                                                                              observed  <2 um   2-5 um >5 um Dome   Deep                              ______________________________________                                         1     8        50      50     0     100     0                                 2    83        76      24     0     95      5                                 4    97        73      27     0     73     27                                 7    120       67      32     0     64     36                                14    54        83      17     0     9      91                                21    20        75      25     0     5      95                                28    15        67      32     0     0      100                               35     9        44      56     0     0      100                               ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Targeted Absorption of 1- to 10-μm Microspheres with Various               Excipients by the Peyer's Patches of the Gut-Associated Lymphoid              Tissues Following Oral Administration                                                                      Absorption by the                                Microsphere Excipient                                                                           Biodegradable                                                                            Peyer's patches                                  ______________________________________                                        Poly(styrene)     No         Very Good                                        Poly(methyl methacrylate)                                                                       No         Very Good                                        Poly(hydroxybutyrate)                                                                           Yes        Very Good                                        Poly(DL-lactide)  Yes        Good                                             Poly(L-lactide)   Yes        Good                                             85:15 Poly(DL-lactide-co-glycolide)                                                             Yes        Good                                             50:50 Poly(DL-lactide-co-glycolide)                                                             Yes        Good                                             Cellulose acetate hydrogen phthalate                                                            No         None                                             Cellulose triacetate                                                                            No         None                                             Ethyl cellulose   No         None                                             ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Primary Anti-Toxin Response to Microencapsulated Versus                       Soluble Staphylococcal Enterotoxoid B                                         Toxoid Dose (μg)     Plasma Anti-Toxin Titer                               Day 20                        Day 10                                          IgM IgG   Form                    IgM  IgG                                    ______________________________________                                        100       Microencapsulated                                                                           1,280  320  1,280                                                                              10,240                               50        Microencapsulated                                                                           640    320  1,280                                                                              5,120                                25        Microencapsulated                                                                           320    <20  640  2,560                                50        Soluble       <20    <20  <20  <20                                  25        Soluble       320    <20  160  <20                                  12.5      Soluble       40     <20  <20  <20                                  ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Secondary Anti-Toxin Response to Microencapsulated Versus                     Soluble Staphylococcal Enterotoxoid B                                                          Plasma Anti-Toxin Titer                                      Toxoid Dose (μg)    Day 10     Day 20                                      per Immunization                                                                        Form         IgM    IgG   IgM  IgG                                  ______________________________________                                        100       Microencapsulated                                                                          320    163,840                                                                             160  81,920                               50        Microencapsulated                                                                          640    81,920                                                                              640  163,840                              25        Microencapsulated                                                                          2,560  40,960                                                                              640  81,920                               50        Soluble      160    <20   80   <20                                  25        Soluble      320    160   160  320                                  12.5      Soluble      160    40    40   80                                   ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Tertiary Anti-Toxin Response to Microencapsulated Versus                      Soluble Staphylococcal Enterotoxoid B                                                          Plasma Anti-Toxin Titer                                      Toxoid Dose (μg)    Day 10     Day 20                                      per Immunization                                                                        Form         IgM    IgG   IgM  IgG                                  ______________________________________                                        100       Microencapsulated                                                                          1,280  655,360                                                                             640  327,680                              50        Microencapsulated                                                                          2,560  327,680                                                                             280  327,680                              25        Microencapsulated                                                                          2,560  327,680                                                                             640  163,840                              50        Soluble      640    1,280 640  640                                  25        Soluble      320    10,240                                                                              80   10,240                               12.5      Soluble      160    1,280 40   1,280                                ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Primary Systemic Anti-Toxin Response Induced by Various                       Parenteral Immunization Routes                                                                         Plasma IgG Anti-Toxin                                Dose (μg) of                                                                             Immunization                                                                             Titer                                                Microencapsulated Toxoid                                                                    Route      Day 15  Day 30                                                                              Day 45                                 ______________________________________                                        100           Intraperitoneal                                                                          12,800  102,400                                                                             204,800                                100           Subcutaneous                                                                              6,400   25,600                                                                             204,800                                ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Secondary Systematic Anti-Toxin Response Induced by                           Various Parenteral Immunization Routes                                        Dose (μg)                                                                  Microencapsulated                                                             Toxoid per  Immunization                                                                             Plasma IgG Anti-Toxin Titer                            Immunization                                                                              Routes     Day 15  Day 30 Day 45                                  ______________________________________                                        100         IP - IP    819,200 1,638,400                                                                            3,276,800                               100         SC - SC    409,600 819,200                                                                              3,276,800                               ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Microspheres Do not Possess Inherent Adjuvant Activity                        Dose             Plasma Anti-Toxin Titer                                      (μg) of       Day 10    Day 20   Day 30                                    Toxoid                                                                              Form       IgM    IgG  IgM  IgG   IgM  IgG                              ______________________________________                                        25    Antigen in 6,400  6,400                                                                              400  12,800                                                                              800  25,600                                 Microspheres                                                            25    Soluble    800    <50  200  800   100  <50                                    Antigen                                                                 25    Antigen plus                                                                             800    <50  200  <50   200  50                                     Placebo                                                                 ______________________________________                                    

                                      TABLE 10                                    __________________________________________________________________________    Systemic Anti-Toxic Response Induced by Parenteral Immunization               μm Microspheres Releasing Antigen at Various Rates                                     Lactide/                                                                           Antigen                                                      Dose (μg)                                                                              Glycolide                                                                          release                                                                           Plasma IgG Anti-Toxin Titer on Day                       of Toxoid                                                                          Form   Ratio                                                                              at 48 Hr                                                                          10 15 20  30  45  60                                     __________________________________________________________________________    100  Soluble                                                                              --   --  <50                                                                              <50                                                                              <50 <50 <50 <50                                    100  Microspheres                                                                         50:50                                                                              60% 400                                                                              -- 6,400                                                                             3,200                                                                             --  --                                     100  Microspheres                                                                         50:50                                                                              30% 400                                                                              -- 12,800                                                                            6,400                                                                             --  --                                     100  Microspheres                                                                         50:50                                                                              10% -- 6,400                                                                            --  102,400                                                                           102,400                                                                           51,200                                 100  Microspheres                                                                         85:15                                                                               0% -- 3,200                                                                            --  51,200                                                                            102,400                                                                           102,400                                __________________________________________________________________________

                  TABLE 11                                                        ______________________________________                                        Results of CPE Inhibition Assays on Serum Samples                             from the JE Vaccine Immunization Studies                                              Dilution of serum capable of                                                  reducing virus-induced CPE by                                                 50% on Day                                                            Animal    21           49      77                                             ______________________________________                                        Group 1 = Untreated Control                                                   GMT.sup.a <10          11      11                                             Average   <10          11      11                                             Maximum   <10          16      <20                                            Minimum   <10          <10     <10                                            Group 2 = 3.0 mg unencapsulated JE vaccine IP on Day 10                       GMT       44           73      50                                             Average   55           95      71                                             Maximum   127          254     160                                            Minimum   <10          13      <10                                            Group 3 = 3.0 mg unencapsulated JE vaccine IP on Days 0, 14 and 42            GMT       507          3,880   1,576                                          Average   934          5,363   2,951                                          Maximum   4,064        >10,240 >10,240                                        Minimum   160          806     254                                            Group 4 = 3.0 mg unencapsulated + 3.0 mg microencapsulated JE                 vaccine IP on Day 0                                                           GMT       77           718     1,341                                          Average   803          1,230   2,468                                          Maximum   320          5,120   10,240                                         Minimum   13           160     254                                            ______________________________________                                         .sup.a GMT = Geometric mean titers.                                      

                  TABLE 12                                                        ______________________________________                                        Results of Plaque-Reduction Assays on Pooled Serum                            Samples from the JE Vaccine Immunization Studies                                               Serum dilution to reach                                      Group Treatment     Day    50% endpoint                                                                           80% endpoint                              ______________________________________                                        .sup. Controls       0     <10      <10                                       1     Controls      14     <10      <10                                       1     Controls      21     <10      <10                                       1     Controls      42     <10      <10                                       1     Controls      49     <10      <10                                       1     Controls      84     <10      <10                                       2     Unencapsulated JE                                                                            0     <10      <10                                        2.sup.b                                                                            Unencapsulated JE                                                                           14     160      20                                        2     Unencapsulated JE                                                                           21     ND.sup.c ND                                        2     Unencapsulated JE                                                                           42     320      80                                        2     Unencapsulated JE                                                                           49     320      40                                        2     Unencapsulated JE                                                                           84     640      160                                        3.sup.d                                                                            Unencapsulated JE                                                                            0     <10      <10                                       3     Unencapsulated JE                                                                           14     160      40                                        3     Unencapsulated JE                                                                           21     2,560    640                                       3     Unencapsulated JE                                                                           42     1,280    640                                       3     Unencapsulated JE                                                                           49     5,120    2,560                                     3     Unencapsulated JE                                                                           84     2,560    1,280                                     .sup. Microencapsulated JE                                                                         0     <10      <10                                       4     Microencapsulated JE                                                                        14     160      20                                        4     Microencapsulated JE                                                                        21     320      80                                        4     Microencapsulated JE                                                                        42     5,120    640                                       4     Microencapsulated JE                                                                        49     5,120    640                                       4     Microencapsulated JE                                                                        84     10,000   2,560                                     ______________________________________                                         .sup.a Untreated controls.                                                    .sup.b Animals received 3.0 mg of unencapsulated JE vaccine IP on Day 0.      .sup.c ND = Not determined (insufficient sample quantity).                    .sup.d Animals received 3.0 mg of unencapsulated JE vaccine IP on Day 0,      14 and 42.                                                                    .sup.e Animals received 3.0 mg of unencapsulated and 3.0 mg of                microencapsulated JE vaccine IP on Day 0.                                

                                      TABLE 13                                    __________________________________________________________________________    The Induction of TNP-Specific Antibodies in the Serum Mucosal Secretions      of BALB/C Mice by Oral Immunization with Microencapsulated TNP-KLH                               ng Immunoglobulin/mL sample                                       Time after                                                                           Biologic                                                                           IgM      IgG       IgA                                     Immunogen                                                                            immunization                                                                         sample                                                                             Total                                                                             Anti-TNP                                                                           Total                                                                              Anit-TNP                                                                           Total                                                                              Anti-TNP                           __________________________________________________________________________    Control                                                                              Day 14 Gut wash                                                                           <1  <1   62   <1   79,355                                                                             25                                               Saliva                                                                             <40 <10  <40  <10  2,651                                                                              <10                                              Serum                                                                              445,121                                                                           6    5,503,726                                                                          37   1,470,553                                                                          32                                 Unencapsulated                                                                       Day 14 Gut wash                                                                           4   1    131  <1   64,985                                                                             17                                 TNP-KLH       Salvia                                                                             <40 <10  <40  <10  1,354                                                                              <10                                              Serum                                                                              298,733                                                                           11   6,000,203                                                                          29   1,321,986                                                                          21                                 TNP-KLH                                                                              Day 14 Gut wash                                                                           3   <1   130  <1   95,368                                                                             222                                Microcapsules Saliva                                                                             <40 <10  <40  <10  1,461                                                                              88                                               Serum                                                                              360,987                                                                           1,461                                                                              5,312,896                                                                          572  1,411,312                                                                          1,077                              Unencapsulated                                                                       Day 28 Gut wash                                                                           <1  <1   94   <1   88,661                                                                             64                                 TNP-KLH       Saliva                                                                             <40 <10  <40  <10  1,278                                                                              <10                                              Serum                                                                              301,223                                                                           21   5,788,813                                                                          67   1,375,322                                                                          63                                 TNP-KLH                                                                              Day 28 Gut wash                                                                           4   <1   122  2    82,869                                                                             422                                Microcapsules Saliva                                                                             <40 <10  <40  <10  1,628                                                                              130                                              Serum                                                                              320,192                                                                           1,904                                                                              5,591,503                                                                          2,219                                                                              1,277,505                                                                          1,198                              __________________________________________________________________________

                  TABLE 14                                                        ______________________________________                                        Plasma IgM and IgG Anti-Toxin Levels on Day 20                                Following Primary, Secondary, and Tertiary Oral Immunization with             Soluble or Microencapsulated (50:50 DL-PLG) Staphylococcal Toxoid                               Plasma anti-toxin titer on day 20                           Enterotoxoid      following oral immunization                                 does (μg) per  Primary   Secondary                                                                             Tertiary                                  immunization                                                                            Form    IgM    IgG  IgM  IgG  IgM  IgG                              ______________________________________                                        100       Micro-  80     1,280                                                                              320  5,120                                                                              1,280                                                                              40,960                                     spheres                                                             100       Soluble <20    <20  80   <20  640  <20                              ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        Toxin-Specific IgA Antibodies in the Saliva and Gut                           Fluids of Mice of Days 10 and 20 After Tertiary Oral Immunization             with Soluble or Microencapsulated Enterotoxoid                                                  IgA anti-enterotoxin titer following                        Enterotoxoid      tertiary oral immunization                                  dose (μg) per  Day 10       Day 20                                         immunization                                                                            Form    Saliva  Gut Wash                                                                             Saliva                                                                              Gut Wash                               ______________________________________                                        100       Micro-  1,280   1,024  640   256                                              spheres                                                             100       Soluble 40      <8     10    <8                                     ______________________________________                                    

                                      TABLE 16                                    __________________________________________________________________________    Serum Anti-Toxin Antibody Levels Induced Through Intratracheal                Immunization with                                                             Soluble or Microencapsulated Staphylococcal Enterotoxin B Toxoid                             Plasma anti-toxin of day following intratracheal                              immunization                                                   Enterotoxoid   10       20        30        40                                dose (μg)                                                                        Form     IgM                                                                              IgG                                                                              IgA                                                                              IgM                                                                              IgG IgA                                                                              IgM                                                                              IgG IgA                                                                              IgM                                                                              IgG IgA                        __________________________________________________________________________    50    Microencapsulated                                                                      <50                                                                              <50                                                                              <50                                                                              200                                                                              25,600                                                                            400                                                                              400                                                                              51,200                                                                            400                                                                              200                                                                              51,200                                                                            400                        50    Soluble  <50                                                                              <50                                                                              <50                                                                              <50                                                                              <50 <50                                                                              <50                                                                              <50 <50                                                                              <50                                                                              <50 <50                        __________________________________________________________________________

                                      TABLE 17                                    __________________________________________________________________________    Bronchial-Alveolar Washing Antibody Levels Induced Through Intratracheal      Immunization with Soluble or Microencapsulated Staphylococcal Enterotoxin     B Toxoid                                                                                     Bronchial-alveolar washing anti-toxin titer                                   on day following intratracheal immunization                    Enterotoxoid   10       20       30       40                                  dose (μg)                                                                        Form     IgM                                                                              IgG                                                                              IgA                                                                              IgM                                                                              IgG                                                                              IgA                                                                              IgM                                                                              IgM                                                                              IgA                                                                              IgM                                                                              IgG                                                                              IgA                           __________________________________________________________________________    50    Microencapsulated                                                                      <5 <5 <5 <5 80 <5 <5 1,280                                                                            320                                                                              <5 1,280                                                                            320                           50    Soluble  <5 <5 <5 <5 <5 <5 <5 <5 <5 <5 20 50                            __________________________________________________________________________

                                      TABLE 18                                    __________________________________________________________________________    Anti-SEB Toxin Antibody Responses Induced in Various Biological Fluids        by Mixed Route Immunization Protocols Using Microencapsulated SEB Toxoid               Dose of                                                              Route of Microencapsulated                                                                      Anti-toxin titer on day 20 following secondary                                immunization                                                Immunization                                                                           SEB toxoid per                                                                         Plasma     Gut Wash  Bronchial Wash                         Primary                                                                           Secondary                                                                          Immunization (μg)                                                                   IgM                                                                              IgG  IgA                                                                              IgM                                                                              IgG IgA                                                                              IgM                                                                              IgG IgA                             __________________________________________________________________________    1P  1P   100      3,200                                                                            1,638,400                                                                          <50                                                                              <20                                                                              10,240                                                                              <20                                                                            <5 10,240                                                                              <5                            1P  Oral 100      1,600                                                                              204,800                                                                          <50                                                                              <20                                                                                640                                                                               640                                                                            <5  2,560                                                                            1,280                           1P  1T   100      1,600                                                                              819,200                                                                          <50                                                                              <20                                                                               2,560                                                                            2,560                                                                            <5 20,480                                                                            2,560                           __________________________________________________________________________

                  TABLE 19                                                        ______________________________________                                        Concentration of Etretinate in Mouse Serum After Oral                         Dosing with Microencapsulated and Unencapsulated Etretinate                             Etretinate Concentration, ng/mL                                     Times/hr    Microcapsules                                                                            Uncapsulated Drug                                      ______________________________________                                        1           4,569      191                                                    3           634        158                                                    6           242        <31                                                    24          ND         ND                                                     ______________________________________                                         ND = None detected                                                       

What is claimed is:
 1. A method of delivering a bioactive agent to themucosally associated lymphoreticular tissues of an animal, comprisingadministering microcapsules to said animal so that an immunogenicallyeffective amount of said microcapsules reach and are selectively takenup by said mucosally associated lymphoreticular tissues, wherein saidmicrocapsules comprise said agent encapsulated in a biocompatibleexcipient and wherein said microcapsules are of a size of betweenapproximately 1 micrometer and approximately 10 micrometers.
 2. Themethod of claim 1, wherein said bioactive agent is a drug, nutrient,immunomodulator, lymphokine, monokine, cytokine, or antigen.
 3. Themethod of claim 1, wherein said bioactive agent is an antigen.
 4. Themethod of claim 3, wherein said antigen is an allergen, viral antigen,bacterial antigen, protozoan antigen, or a fungal antigen.
 5. The methodof claim 3, wherein said antigen is an influenzae antigen,Staphylococcus antigen, respiratory syncytial antigen, parainfluenzavirus antigen, Hemophilus influenza antigen, Bordetella pertussisantigen, Neisseria gonorrhoea antigen, Streptococcus pneumoniae antigen,Plasmodium falciparum antigen, helminthic pathogen antigen, or anantigen to vaccinate against allergies.
 6. The method of claim 3,wherein said antigen is an influenza virus or staphylococcal enterotoxinB.
 7. The method of claim 3, wherein said bioactive agent furtherincludes a cytokine.
 8. The method of claim 7, wherein said bioactiveagent further includes an adjuvant.
 9. The method of claim 1, whereinsaid bioactive agent is a mixture of a cytokine and an adjuvant.
 10. Themethod of claim 1, wherein said bioactive agent comprises a peptide,protein or nucleic acid.
 11. The method of claim 1, wherein saidadministering is by oral, nasal, rectal, vaginal, ophthalmical, or oralinhalation administration.
 12. The method of claim 1, wherein saidbiocompatible excipient is a poly(lactide-co-glycolide), poly(lactide),poly(glycolide), copolyoxalate, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramide), polyorthoester,poly(β-hydroxybutyric acid), polyanhydride, or a mixture thereof. 13.The method of claim 1, wherein said microcapsules comprise a pluralityof first microcapsules having a size of between approximately 1micrometer and approximately 5 micrometers and a plurality of secondmicrocapsules having a size of between approximately 5 micrometers andapproximately 10 micrometers, and wherein said administering comprisesthe delivery of a mixture of said first and second microcapsules to saidanimal to provide both systemic immunity and mucosal immunity.
 14. Themethod of claim 1, wherein the delivery is orally and the mucosallyassociated lymphoeticular tissues are the Peyer's patch.
 15. A method ofdelivering a bioactive agent to the mucosally associated lymphoreticulartissues of an animal, comprising administering microcapsules to saidanimal so that an immunogenically effective amount of said microcapsulesreach and are selectively taken up by said mucosally associatedlymphoreticular tissues, wherein said microcapsules comprise said agentencapsulated in a biocompatible excipient and wherein said microcapsulesare of a size of less than approximately 10 micrometers.
 16. The methodof claim 15, wherein the delivery is orally and the mucosally associatedlymphoeticular tissues are the Peyer's patch.
 17. A method of providingsystemic immunity in an animal, comprising administering microcapsulesto said animal so that immunogenically effective amount of saidmicrocapsules reach and are selectively taken up by themucosally-associated lymphoreticular tissues to provide a systemicimmune response in said animal, wherein said microcapsules comprise abioactive agent encapsulated in a biocompatible excipient and whereinsaid microcapsules are of a size of between approximately 1 micrometerand approximately 5 micrometers.
 18. The method of claim 17, whereinsaid bioactive agent is an immunomodulator, lymphokine, monokine,cytokine, or antigen.
 19. The method of claim 17, wherein said bioactiveagent is an antigen.
 20. The method of claim 19, wherein said antigen isan allergen, viral antigen, bacterial antigen, protozoan antigen, or afungal antigen.
 21. The method of claim 19, wherein said antigen is aninfluenzae antigen, Staphylococcus antigen, respiratory syncytialantigen, parainfluenza virus antigen, Hemophilus influenza antigen,Bordetella pertussis antigen, Neisseria gonorrhoea antigen,Streptococcus pneumoniae antigen, Plasmodium falciparum antigen,helminthic pathogen antigen, or an antigen to vaccinate againstallergies.
 22. The method of claim 19, wherein said antigen is aninfluenza virus or staphylococcal enterotoxin B.
 23. The method of claim19, wherein said bioactive agent further includes a cytokine.
 24. Themethod of claim 23, wherein said bioactive agent further includes anadjuvant.
 25. The method of claim 17, wherein said bioactive agent is amixture of a cytokine and an adjuvant.
 26. The method of claim 17,wherein said bioactive agent comprises a peptide, protein or nucleicacid.
 27. The method of claim 17, wherein said administering is by oral,nasal, rectal, vaginal, ophthalmical, or oral inhalation administration.28. The method of claim 17, wherein said biocompatible excipient is apoly(lactide-co-glycolide), poly(lactide), poly(glycolide),copolyoxalate, polycaprolactone, poly(lactide-co-caprolactone),poly(esteramide), polyorthoester, poly(β-hydroxybutyric acid),polyanhydride, or a mixture thereof.
 29. A method of providing mucosalimmunity in an animal, comprising administering microcapsules to saidanimal so that an immunogenically effective amount of said microcapsulesreach and are selectively taken up by the mucosally-associatedlymphoreticular tissues to provide a mucosal immune response in saidanimal, wherein said microcapsules comprise a bioactive agentencapsulated in a biocompatible excipient and wherein said microcapsulesare of a size of between approximately 5 micrometers and approximately10 micrometers.
 30. The method of claim 29, wherein said bioactive agentis an immunomodulator, lymphokine, monokine, cytokine, or antigen. 31.The method of claim 29, wherein said bioactive agent is an antigen. 32.The method of claim 31, wherein said antigen is an allergen, viralantigen, bacterial antigen, protozoan antigen, or a fungal antigen. 33.The method of claim 31, wherein said antigen is an influenzae antigen,Staphylococcus antigen, respiratory syncytial antigen, parainfluenzavirus antigen, Hemophilus influenza antigen, Bordetella pertussisantigen, Neisseria gonorrhoea antigen, Streptococcus pneumoniae antigen,Plasmodium falciparum antigen, helminthic pathogen antigen, or anantigen to vaccinate against allergies.
 34. The method of claim 31,wherein said antigen is an influenza virus or staphylococcal enterotoxinB.
 35. The method of claim 31, wherein said bioactive agent furtherincludes a cytokine.
 36. The method of claim 35, wherein said bioactiveagent further includes an adjuvant.
 37. The method of claim 29, whereinsaid bioactive agent is a mixture of a cytokine and an adjuvant.
 38. Themethod of claim 29, wherein said bioactive agent comprises a peptide,protein or nucleic acid.
 39. The method of claim 29, wherein saidadministering is by oral, nasal, rectal, vaginal, ophthalmical, or oralinhalation administration.
 40. The method of claim 29, wherein saidbiocompatible excipient is a poly(lactide-co-glycolide), poly(lactide),poly(glycolide), copolyoxalate, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramide), polyorthoester,poly(β-hydroxybutyric acid), polyanhydride, or a mixture thereof.
 41. Amethod of providing mucosal immunity in an animal, comprisingadministering effective amounts of microcapsules to said animal so thatan immunogenically effective amount of said microcapsules reach and areselectively taken up by the mucosally-associated lymphoreticular tissuesto provide a mucosal immune response in said animal, wherein saidmicrocapsules comprise effective amounts of a bioactive agentencapsulated in a biocompatible excipient and wherein said microcapsulesare of a size of between approximately 1 micrometer and approximately 5micrometers.
 42. The method of claim 41, wherein said bioactive agent isan immunomodulator, lymphokine, monokine, cytokine, or antigen.
 43. Themethod of claim 41, wherein said bioactive agent is an antigen.
 44. Themethod of claim 43, wherein said antigen is an allergen, viral antigen,bacterial antigen, protozoan antigen, or a fungal antigen.
 45. Themethod of claim 43, wherein said antigen is an influenzae antigen,Staphylococcus antigen, respiratory syncytial antigen, parainfluenzavirus antigen, Hemophilus influenza antigen, Bordetella pertussisantigen, Neisseria gonorrhoea antigen, Streptococcus pneumoniae antigen,Plasmodium falciparum antigen, helminthic pathogen antigen, or anantigen to vaccinate against allergies.
 46. The method of claim 43,wherein said antigen is an influenza virus or staphylococcal enterotoxinB.
 47. The method of claim 43, wherein said bioactive agent furtherincludes a cytokine.
 48. The method of claim 47, wherein said bioactiveagent further includes an adjuvant.
 49. The method of claim 41, whereinsaid bioactive agent is a mixture of a cytokine and an adjuvant.
 50. Themethod of claim 41, wherein said bioactive agent comprises a peptide,protein, or nucleic acid.
 51. The method of claim 41, wherein saidadministering is by oral, nasal, rectal, vaginal, ophthalmical, or oralinhalation administration.
 52. The method of claim 41, wherein saidbiocompatible excipient is a poly(lactide-co-glycolide), poly(lactide),poly(glycolide), copolyoxalate, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramide), polyorthoester,poly(β-hydroxybutyric acid), polyanhydride, or a mixture thereof.